In vivo modulation of neuronal transport

ABSTRACT

A hybrid protein (GFP-TTC) comprising the non-toxic proteolytic C fragment of tetanus toxin fused to green fluorescent protein was used to analyze the functional synaptic organization of neural networks. When injected intramuscularly in vivo, the GFP-TTC hybrid protein binds to tetanus neurotoxin receptors and clusters very rapidly to the active neuromuscular junction. Membrane traffic by GFP-TTC at the pre-synaptic level of the neuromuscular junction is strongly and rapidly influenced by exogenously co-injecting neurotrophic factors, such as BDNF, NT-4, and GDNF, but not by NGF, NT-3, and CNTF. The membrane traffic, directly detected using GFP-TTC in vivo, permits methods of analyzing synaptic functioning as well as methods of modulating neuronal transport using neurotrophic factors and agonists or antagonists thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of copendingU.S. application Ser. No. 09/816,467, filed Mar. 26, 2001, which is acontinuation of U.S. application Ser. No. 09/129,368, filed Aug. 5,1998, now abandoned, which claims the benefit of Provisional ApplicationNo. 60/055,615, filed Aug. 14, 1997 and Provisional Application No.60/065,236, filed Nov. 13, 1997. The entire disclosure of each of theseapplications is relied upon and incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] This invention relates to the use of part of tetanus toxin fordelivering a composition to the central nervous system of a human oranimal. This invention also relates to a hybrid fragment of tetanustoxin, a polynucleotide that hybridizes with natural tetanus toxin, anda composition containing the tetanus toxin fragment as an activemolecule. Further, this invention relates to a vector comprising apromoter and a nucleic acid sequence encoding the tetanus toxinfragment.

[0003] Tetanus toxin is produced by Clostridium tetani as an inactive,single, polypeptide chain of 150 kD composed of three 50 kD domainsconnected by protease-sensitive loops. The toxin is activated uponselective proteolytic cleavage, which generates two disulfide-linkedchains: L (light, 50 kD) and H (heavy, 100 kD) [Montecucco C. andSchiavo G. Q. Rev. Biophys., (1995), 28: 423-472].

[0004] Evidence for the retrograde axonal transport of tetanus toxin tocentral nervous system (CNS) has been described by Erdmann et al.[Naunyn Schmiedebergs Arch Phamacol., (1975), 290:357-373], Price et al.[Science, (1975), 188:945-94], and Stoeckel et al. [Brain Res., (1975),99:1-16]. In each of these studies, radiolabeled toxin was found insidemembrane bound vesicles. Another property was the transynaptic movementof tetanus toxin that was demonstrated first by autoradiographiclocalization of ¹²⁵I-labeled tetanus toxin in spinal cord interneuronsafter injection into a muscle [Schwab and Thoenen, Brain Res., (1976),105:218-227].

[0005] The structure of this tetanus toxin has been elucidated byHelting et al. [J. Biol. Chem., (1977), 252:187-193]. Papain cleaves thetetanus toxin in two fragments:

[0006] the C terminal part of the heavy chain, 451 amino acids, alsocalled fragment C; and

[0007] the other part contained the complementary portion calledfragment B linked to the light chain (fragment A) via a disulfide bond.

[0008] European Patent No. EP 0 030 496 B1 showed the retrogradetransport of a fragment B-II_(b) to the CNS and was detected afterinjection in the median muscle of the eye in primary and second orderneurons. This fragment may consist of “isofragments” obtained byclostridial proteolysis. Later, this fragment B-II_(b) was demonstratedto be identical to fragment C obtained by papain digestion by Eisel etal. [EMBO J., 1986, 5:2495-2502].

[0009] This EP patent also demonstrated the retrograde transport of aconjugate consisting of a I_(bc) tetanus toxin fragment coupled by adisulfide bond to B-II_(b) from axonal endings within the muscle to themotoneuronal perikarya and pericellular spaces. (The I_(bc) fragmentcorresponds to the other part obtained by papain digestion as describedabove by Helting et al.). There is no evidence that this conjugate wasfound in second order neurons. The authors indicated that a conjugateconsisting of the fragment B-II_(b) coupled by a disulfide bond to atherapeutic agent was capable of specific fixation to gangliosides andsynaptic membranes. No result showed any retrograde axonal transport ora transynaptic transport for such conjugate.

[0010] Another European Patent, No. EP 0 057 140 B1, showed equally theretrograde transport of a fragment II_(c) to the CNS. As in the EuropeanPatent No. EP 0 030 496 B1, the authors indicated that a conjugateconsisting of the fragment II_(c) and a therapeutic agent was capable ofspecific fixation, but no result illustrated such allegation. Thisfragment II_(c) corresponds to the now called fragment C obtained bypapain digestion.

[0011] Francis et al. [J. Biol. Chem., (1995), 270(25):15434-15442] ledan in vitro study showing the internalization by neurons of hybridbetween SOD-1 (Cu Zn superoxide dismutase) and a recombinant C tetanustoxin fragment by genetic recombination. This recombinant C tetanustoxin fragment was obtained from Halpern group. (See ref. 11).

[0012] Moreover, Kuypers H. G. J. M and Ugolini G. [TINS, (1990),13(2):71-75] indicated in their publication concerning viruses astransneuronal tracers that, despite the fact that tetanus toxin fragmentbinds to specific receptors on neuronal membranes, transneuronallabeling is relatively weak and can be detected only in some of thesynaptically connected neurons.

[0013] Notwithstanding these advances in the art, there still exists aneed for methods for delivering compositions into the human or animalcentral nervous system. There also exists a need in the art forbiological agents that can achieve this result.

[0014] Additionally, activity-dependent modification of neuronalconnectivity and synaptic plasticity play an important role in thedevelopment and function of the nervous system. Recently, much efforthas been dedicated to following such modifications by the engineering ofnew optically detectable genetic tools. For example, fused to a reportergene such as LacZ or GFP (Green Fluorescent Protein), the atoxicC-terminal fragment of tetanus toxin (or TTC fragment) can trafficretrogradely and transsynaptically inside a restricted neural networkeither after direct injection of the hybrid protein (Coen et al., 1997),or when expressed as a transgene in mice (Maskos et al., 2002). Thedynamics of βgal-TTC clustering at the neuromuscular junction (NMJ) isstrongly dependent on a presynaptic neuronal activity and probablyinvolves fast endocytic pathways (Miana-Mena et al., 2002). Neuronalactivity may induce this clustering and internalization at the NMJ byenhancing the secretion and/or action of various molecules at thesynapse.

[0015] Over the past decade, various data indicate that neurotrophins, afamily of structurally and functionally related proteins, including NGF(Nerve Growth Factor); BDNF (Brain Derived Neurotrophic Factor);Neurotrophin 3 (NT-3) and Neurotrophin 4 (NT-4), not only promoteneuronal survival and morphological differentiation, but also canacutely modify synaptic transmission and connectivity in centralsynapses, thus providing a connection between neuronal activity andsynaptic plasticity (McAllister et al., 1999; Poo, 2001; Tao and Poo,2001). The role of these factors in neurotransmission betweenmotoneurons and skeletal muscle cells has been studied using Xenopusnerve-muscle co-culture studies, whereby the treatment of these cultureswith exogenous BDNF, NT-3 or NT-4 leads to an increase of synaptictransmission by enhancing neurotransmitter secretion (Lohof et al.,1993; Stoop and Poo, 1996; Wang and Poo, 1997). Moreover, the muscularexpression of NT-3 and NT-4 (Funakoshi et al., 1995; Xie et al., 1997),as well as NT-4 secretion (Wang and Poo, 1997) are regulated byelectrical activity. This family of proteins thus provides a molecularlink between electrical neuronal activity and synaptic changes.

[0016] The cellular actions of neurotrophins are mediated by two typesof receptors: the p75^(NTR) receptor, mainly expressed during earlyneuronal development, and a Trk tyrosine kinase receptor (Bothwell,1995). The interaction of neurotrophins with Trk receptors is specific.TrkB and TrkC, are activated by BDNF/NT-4 and NT-3, respectively, andare expressed by motor neurons. TrkA, which is expressed by sensoryneurons, is activated by NGF. Recently, evidence for a co-traffickingbetween TTC and the neurotrophin receptor p75^(NTR) has been reported incultured motoneurons (Lalli and Schiavo, 2002), as well as theactivation by tetanus toxin and the TTC fragment of intracellularpathways involving Trk receptors in cultured cortical neurons (Gil etal., 2003).

[0017] Notwithstanding the knowledge in the art, there still exists aneed for understanding the influences of neurotrophins and otherneurotrophic factors on TTC traffic at the NMJ in vivo and fordeveloping methods of using these neurotrophins and neurotrophicfactors, and agonists or antagonists thereof, to modulate the neuronaltransport of a tetanus toxin or a fusion protein comprising a fragment Cof the tetanus toxin.

SUMMARY OF THE INVENTION

[0018] This invention aids in fulfilling these needs in the art. Moreparticularly, this invention provides a method for in vivo delivery ofdesired composition into the central nervous system (CNS) of the mammal,wherein the composition comprises a non-toxic proteolytic fragment oftetanus toxin (TT) in association with at least a molecule having abiological function. The composition is capable of in vivo retrogradetransport and transynaptic transport into the CNS and of being deliveredto different areas of the CNS.

[0019] This invention also provides a hybrid fragment of tetanus toxincomprising fragment C and fragment B or a fraction thereof of at least11 amino acid residues or a hybrid fragment of tetanus toxin comprisingfragment C and fragment B or a fraction thereof of at least 11 aminoacid residues and a fraction of fragment A devoid of its toxic activitycorresponding to the proteolytic domain having a Zinc-binding motiflocated in the central part of the chain between the amino acids 225 and245, capable of transferring in vivo a protein, a peptide, or apolynucleotide through a neuromuscular junction and at least onesynapse.

[0020] Further, this invention provides a composition comprising anactive molecule in association with the hybrid fragment of tetanus toxin(TT) or a variant thereof. The composition is useful for the treatmentof a patient or an animal affected with CNS disease, which comprisesdelivering a composition of the invention to the patient or animal. Inaddition, the composition of this invention may be useful to elicit aimmune response in the patient or animal affected with CNS, whichcomprises delivering a composition of the invention to the patient oranimal.

[0021] Moreover, this invention provides polynucleotide variantfragments capable of hybridizing under stringent conditions with thenatural tetanus toxin sequence. The stringent conditions are for exampleas follows: at 42° C. for 4 to 6 hours in the presence of 6×SSC buffer,1× Denhardt's Solution, 1% SDS, and 250 μg/ml of tRNA. (1×SSCcorresponds to 0.15 M NaCl and 0.05 M sodium citrate; 1× Denhardt'ssolution corresponds to 0.02% Ficoll, 0.02% polyvinyl pyrrolidone and0.02% bovine serum albumin). The two wash steps are performed at roomtemperature in the presence of 0.1×SCC and 0.1% SDS.

[0022] A polynucleotide variant fragment means a polynucleotide encodingfor a tetanus toxin sequence derived from the native tetanus toxinsequence and having the same properties of transport.

[0023] In addition, the invention provides a vector comprising apromoter capable of expression in muscle cells and optionally anenhancer, a nucleic acid sequence coding for the fragment of tetanustoxin of the invention or an amino acid variant fragment of theinvention associated with a polynucleotide coding for a protein or apolypeptide of interest. In a preferred embodiment of the invention thepromoter can be the CMV promoter and preferably the CMV promotercontained in pcDNA 3.1 (In Vitrogen, ref. V790-20), or the promoter βactin as described in Bronson S. V. et al. (PNAS, 1996, 93:9067-9072).

[0024] In addition, the invention provides a vector comprising apromoter capable of expression in neuronal cells or in precursors (suchNT2(hNT) precursor cells from Stratagene reference # 204101) andoptionally an enhancer, a nucleic acid sequence coding for the fragmentof tetanus toxin of the invention or an amino acid variant fragment ofthe invention associated with a polynucleotide coding for a protein or apolypeptide of interest. In a preferred embodiment of the invention thepromoter can be β actin (see the above reference). These vectors areuseful for the treatment of a patient or an animal infected with CNSdisease comprising delivering the vector of the invention to the patientor animal. In addition, these vectors are useful for eliciting immuneresponses in the patient or animal.

[0025] One advantage of the present invention comprising the fragment oftetanus toxin (fragment A, B, and C) is to obtain a better transport ofthe fragment inside the organism compared with fragment C. Anotheradvantage of the composition of the invention is to obtain a welldefined amino acid sequence and not a multimeric composition. Thus, onecan easily manipulate this composition in gene therapy.

[0026] In another embodiment, this invention provides a method ofmodulating the transport in a neuron of a neurotoxin, such as thetetanus toxin, or a fusion protein comprising a fragment C of thetetanus toxin. These methods comprise administering neurotrophic factorssuch as BDNF, NT-4, and GDNF, and agonists and antagonists thereof, tomodulate internalization at a neuromuscular junction of a neurotoxin ora fusion protein comprising the TTC fragment according to the invention.

[0027] In one embodiment, these methods further comprise administeringto the neuron a TrkB receptor agonist or a TrkB receptor antagonist inan amount sufficient to modulate the neuronal transport of the tetanustoxin or the fusion protein. The term “modulate” and its cognates referto the capability of a compound acting as either an agonist or anantagonist of a certain reaction or activity. The term modulate,therefore, encompasses the terms “increase” and “decrease.” The term“increase,” for example, refers to an increase in the neuronal transportof a polypeptide in the presence of a modulatory compound, relative tothe transport of the polypeptide in the absence of the same compound.Analogously, the term “decrease” refers to a decrease in the theneuronal transport of a polypeptide in the presence of a modulatorycompound, relative to the transport of the polypeptide in the absence ofthe same compound. The neuronal transport of polypeptides can bemeasured as decribed herein or by techniques generally known in the art.

[0028] The TrkB receptor agonists include neurotrophic factors thatactivate a TrkB receptor, such as a Brain Derivated Neurotrophic Factoror a Neurotrophin 4. The TrkB receptor agonists can also includeantibodies that bind to TrkB receptors and activate them. These methodsof using TrkB receptor agonists provide useful methods for enhancing theneuronal transport of a tetanus toxin or a tetanus toxin fusion protein.

[0029] The TrkB receptor antagonists include antibodies that bind to aTrkB receptor agonist, such as those described above, and therebydecrease the activation of a TrkB receptor. For example, theseantibodies can be directed to neurotrophic factors that activate a TrkBreceptor, such as a Brain Derivated Neurotrophic Factor or aNeurotrophin 4. In addition, TrkB receptor antagonists includeantibodies that bind to TrkB receptors and inactivate them. Thesemethods of using TrkB receptor agonists provide useful methods fordecreasing or preventing the neuronal transport of a tetanus toxin or atetanus toxin fusion protein.

[0030] In another embodiment, these methods further compriseadministering to the neuron a GFRα/cRET receptor agonist or a GFRα/cRETreceptor antagonist in an amount sufficient to modulate the neuronaltransport of the tetanus toxin or the fusion protein.

[0031] The GFRα/cRET receptor agonists include neurotrophic factors thatactivate a GFRα/cRET receptor, such as a Glial-Derived NeurotrophicFactor. The GFRα/cRET receptor agonists can also include antibodies thatbind to GFRα/cRET receptors and activate them. These methods of usingGFRα/cRET receptor agonists provide useful methods for enhancing theneuronal transport of a tetanus toxin or a tetanus toxin fusion protein.

[0032] The GFRα/cRET receptor antagonists include antibodies that bindto a GFRα/cRET receptor agonist, such as those described above, andthereby decrease the activation of a GFRα/cRET receptor. For example,these antibodies can be directed to neurotrophic factors that activate aGFRα/cRET receptor, such as a Glial-Derived Neurotrophic Factor. Inaddition, GFRα/cRET receptor antagonists include antibodies that bind toGFRα/cRET receptors and inactivate them. These methods of usingGFRα/cRET receptor agonists provide useful methods for decreasing orpreventing the neuronal transport of a tetanus toxin or a tetanus toxinfusion protein.

[0033] In these methods, the agonist or antagonist can be administeredto neuronal cells that already contain a tetanus toxin or a fusionprotein. Alternatively, the tetanus toxin or fusion protein can beadministered concurrently with or after the administration of theagonist or antagonist.

[0034] In one embodiment, the TTC-containing fusion proteins of thepresent invention comprises a second protein that is encoded by areporter gene, such as the lac Z gene or the Green Fluorescent Proteingene. Such fusion proteins are useful for visualizing modulation of thesynaptic plasticity in vivo, including in a human, for example bymagnetic resonance imaging. For example, the fusion proteins can be usedto monitor and detect the effects of a compound, such as a neurotrophicfactor, on neuronal transport. In these methods, the compound and thefusion protein are administered to a neuron, and the fusion protein isdetected to determine the effect of the compound on the neuronaltransport. In addition, the fusion proteins can be used to detectmodifications in trafficking patterns within a restricted neuralnetwork, such as those used in known animal models for neurodegenerativediseases. The fusion proteins can also be used in screening methods todetect compounds that reduce or prevent neuronal transport of a tetanustoxin. Compounds so identified can be used to prevent or treat tetanusinfections.

[0035] The TTC fragment can also be coupled to a neurotrophic factor andadministered to a patient to treat CNS pathologies associated withproduction defects of different factors. The TTC fragment could also beused as a vector for modulating interactions with proteins involved inneurodegenerative diseases.

[0036] The present invention also provides compositions comprising aTrkB receptor agonist or a GFRα/cRET receptor agonist and a fusionprotein comprising a fragment C of the tetanus toxin fused to a secondprotein. In one embodiment, the TrkB agonist is a neurotrophic factorsuch as a Brain Derivated Neurotrophic Factor or a Neurotrophin 4. Inanother embodiment, the GFRα/cRET receptor agonist is a neurotrophicfactor, such as Glial-Derived Neurotrophic Factor

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] This invention will be more fully described with reference to thedrawings in which:

[0038]FIG. 1 shows the DNA sequence and amino acid sequence of the TTCfragment cloned in PBS:TTC.

[0039]FIG. 2 shows the details of construct PBS:TTC, which is furtherdescribed in Example 1.

[0040]FIG. 3 depicts pGEX:lacZ-TTC construct.

[0041]FIG. 4 shows construct pGEX:TTC-lacZ.

[0042]FIG. 5 depicts the details of the construct pCMV:lacZ-TTC.

[0043]FIG. 6 shows the confocal immunofluorescence analysis of GFP-TTCmembrane traffic at mature mouse LAL NMJs.

[0044] (A) Two hours after the subcutanous injection of GFP-TTC in thevicinity of the LAL muscle, the probe (green) was concentrated at motornerve endings of NMJ. Associated intramuscular motor axons wereimmunostained (red) with an antibody against NF200. GFP-TTC labeling wasalso detected in sensory nerve fibers (arrows) and at the nodes ofRanvier of myelinated axons (arrowheads). (B) Strong nodal labeling withGFP-TTC (green) (arrow) in a single living myelinated axon. Myelin waspassively stained with RH414 dye (red). (C) Two hours after injection asin A, LAL muscle fibers were fixed and labeled for troponin T byindirect immunofluorescence. (C′ and C″) Inset is a side view image of aNMJ showing that GFP-TTC staining (green) is located presynaptically.(D-G) LAL was harvested at various times after GFP-TTC injection and NMJidentified in red by labeling with TRITC-a-BTX (D′-G′). D-D′: 5 minE-E′: 30 min; F-F′: 2 h and G-G′: 24 h.

[0045] Scale bars: A, 20 μm; B, 8 μm; C: 20 μm; D, 2 μm; E-G, 5 μm.

[0046]FIG. 7 shows that BDNF increases GFP-TTC recruitment at the NMJ ina dose-dependent manner.

[0047] (A1-A6) The NMJ on LAL muscles was identified by TRITC-a-BTXlabeling 30 min after in vivo co-injection of GFP-TTC with variousamounts of BDNF. The level of GFP fluorescence was quantified over theseareas (see B). An enhancement of axonal labeling (arrows), moreprononced with higher BDNF concentration, was also detected. Scale bars:20 μm (B) Confocal sections of the NMJ were collected for analysis andprojections generated. TRIT-a-BTX labeling determines the area of theNMJ over which the global GFP fluorescent signal was measured. For each,(n=15-20), the GFP fluorescence was divided by NMJ area (in μm²) toobtain the fluorescence level. Error bars indicate S.D. ** P<0.005;t-test, vs control.

[0048]FIG. 8 depicts the immunofluorescence visualization of TrkB at theLAL NMJ. Two hours after GFP-TTC injection in LAL, confocal analysis wasperformed. (A-B) The fusion protein was identified in green directly byGFP fluorescence. (C-D) TrkB, identified (in red) by indirectimmunofluorescence (see material and methods), was located at the NMJ.(E-F) However, when the two projections were overlaid, no overlap wasfound between the TrkB and the GFP-TTC signals.

[0049] Scale bar: Top: 5 μm; Bottom: 2 μm

[0050]FIG. 9 represents the results of experiments elucidating themechanisms of GFP-TTC recruitment to the NMJ.

[0051] (A) Quantification of GFP-TTC fluorescence was performed, asdescribed in FIG. 7 at various time after co-injection with or without50 ng BDNF.

[0052] (B) After in vitro loading for 45 min with GFP-TTC, the excisedLAL muscle was fixed and SV2 protein detected by indirectimmunofluorescence (red). SV2 labeling was mostly diffuse andconcentrated in a few areas of the NMJ (arrows). Colocalization of SV2with GFP-TTC staining was only observed in a very limited number ofareas. Scale bar: 8 μm.

[0053] (C) Treatment with botulinum type-A neurotoxin to block synapticvesicles exocytosis and endocytosis. 48 hours after BoTx/A injection (asdescribed in material and methods), GFP-TTC, associated or not with 50ng BDNF, was injected in LAL muscle and GFP fluorescence quantified aspreviously. ** P<0.005; t-test, vs control; * P<0.005; t-test vs BoTx/Atreatment.

[0054] (D) Comparison of KCl induced depolarization and BDNF effects onGFP-TTC localization at the NMJ.

[0055]FIG. 10 depicts the localization of GFP-TTC probe in lipidmicrodomains.

[0056] (A) 2 hours after intramuscular injection, GFP-TTC was found indetergent resistant membranes (DRMs) (lanes 4-6) isolated fromgastrocnemius muscle, which also contained the raft marker caveolin-3. Asmall amount of GFP-TTC was also detected in the soluble fraction (lane12).

[0057] (B) GFP-TTC colocalized with the raft marker GM1 at the NMJ. NMJwere identified by Alexa 647-a-BTX binding (in blue). Whereas GFP-TTCwas detected in less than 5 min at the NMJ, CT-b requires 3-5 hours. Atthis time, a diffuse staining which colocalized with the similar GFP-TTClabeling, was obtained, while a few patches labeling only for CT-b werealso observed.

[0058] (C) Intensity profiles of GFP-TTC (green) and Alexa 594labeled-CT-b (red) were performed 5 or 24 h after intramuscularco-injection of both probes in gastrocnemius.

[0059] Scale bar: 5 μm.

[0060]FIG. 11 shows a comparison of GFP-TTC and CT-b localization inmotoneuron cell bodies. Twenty four hours after β-gal-TTC (A) or GFP-TTCand CT-b (B-E) intramuscular injection in gastrocnemius muscle, micewere perfused intracardially and their spinal cords removed. (A) X-galreaction on spinal cord transerve sections showed labeling in motoneuroncell bodies but also in neurites (inset). (B) GFP-TTC and CT-b weredetected on longitudinal section of spinal cord in a significant numberof motoneurons. (C) Probes were detected in vesicles highly concentratedin cell bodies but also in neurites. (D) In neuronal extensions, GFP-TTCand CT-b were detected in different vesicular-like structures. Note thatonly few of them were positive for both probes. (E) Note that neitherGFP-TTC nor CT-b were detected in the nucleus as shown in one opticalsection.

[0061] Scale bars: A, 0.2 mm; inset, 50 μm; B, 20 μm; C, 10 μm; D, 5 μm;E, 2 μm.

DETAILED DESCRIPTION

[0062] Tetanus toxin is a potent neurotoxin of 1315 amino acids that isproduced by Clostridium tetani (1, 2). It prevents the inhibitoryneurotransmitter release from spinal cord interneurons by a specificmechanism of cell intoxication (for review see ref 3). This pathologicalmechanism has been demonstrated to involve retrograde axonal andtransynaptic transport of the tetanus toxin. The toxin is taken up bynerve endings at the neuromuscular junction, but does not act at thissite; rather, the toxin is transported into a vesicular compartment andtravels along motor axons for a considerable distance until it reachesits targets. The transynaptic movement of tetanus toxin was firstdemonstrated by autoradiographic localization in spinal cordinterneurons after injection into a muscle (4). However, previousstudies of transynaptic passage of tetanus toxin from motoneurons werelimited by the rapid development of clinical tetanus and death of theexperimental animal (4, 5, 6).

[0063] A fragment of tetanus toxin obtained by protease digestion, the Cfragment, has been shown to be transported by neurons in a similarmanner to that of the native toxin without causing clinical symptoms (7,8, 9, 10). A recombinant C fragment was reported to possess the sameproperties as the fragment obtained by protease digestion (11). The factthat an atoxic fragment of the toxin molecule was able to migrateretrogradely within the axons and to accumulate into the CNS led tospeculation that such a fragment could be used as a neurotrophic carrier(12). A C fragment chemically conjugated to various large proteins wastaken up by neurons in tissue culture (13) and by motor neurons inanimal models (ref. 12, 14, and 15). According to the invention thefragment of tetanus toxin consists of a non-toxic proteolytic fragmentof tetanus toxin (TT) comprising a fragment C and a fragment B or afraction thereof of at least 11 amino acid residues or a non-toxicproteolytic fragment of tetanus toxin (TT) comprising a fragment C and afragment B or a fraction thereof of at least 11 amino acids residues anda fraction of a fragment A devoid of its toxic activity corresponding tothe proteolytic domain having a zinc-binding motif located in thecentral part of the chain between the amino acids 225 and 245 (cf.Montecucco C. and Schiavo G. Q. Rev. Biophys., (1995), 28:423472). Thusthe fraction of the fragment A comprises, for example, the amino acidsequence 1 to 225 or the amino acid sequence 245 to 457, or the aminoacid sequence 1 to 225 associated with amino acid sequence 245 to 457.

[0064] The molecule having a biological function is selected from thegroup consisting of protein of interest, for example, for thecompensation or the modulation of the functions under the control of theCNS or the spinal cord or the modulation of the functions in the CNS orthe spinal cord, or protein of interest to be delivered by gene therapyexpression system to the CNS or the spinal cord. The proteins ofinterest are, for example, the protein SMN implicated in spinal muscularatrophy (Lefebvre et al., Cell, (1995), 80:155-165 and Roy et al., Cell,(1955), 80:167-178); neurotrophic factors, such as BDNF (Brain-derivedneurotrophic factor); NT-3 (Neurotrophin-3); NT4/5; GDNF (Glialcell-line-derived neurotrophic factor); IGF (Insulin-like growth factor)(Oppenheim, Neuron, (1996), 17:195-197; Thoenen et al., Exp. Neurol.,(1933), 124:47-55 and Henderson et al., Adv. Neurol., (1995),68:235-240); or PNI (protease nexin I) promoting neurite outgrowth (thisfactor can be used for the treatment of Alzheimer disease (Houenou etal., PNAS, (1995), 92:895-899)); or SPI3 a serine protease inhibitorprotein (Safaei, Dev. Brain Res., (1997), 100: 5-12); or ICE(Interleukin-1β converting Enzyme) a factor implicated in apoptosis, toavoid apoptosis (Nagata, Cell, (1997), 88:355-365); or Bcl-2, a keyintracellular regulator of programmed cell death (Jacobson, M. D.(1997), Current Biology, 7:R277-R281); or green fluorescent protein(Lang et al., Neuron, (1997), 18:857-863) as a marker; enzyme (ex:β-Gal); endonuclease like 1-SceI (Choulika A., et al. (1995), Molecularand Cellular biology, 15 (4):1968-1973 or CRE (Gu H., et al. (1994),Science, 265:103-106); specific antibodies; drugs specifically directedagainst neurodegenerative diseases such as latero spinal amyotrophy.Several molecules can be associated with a TT fragment.

[0065] In association means an association obtained by geneticrecombination. This association can be realized upstream as well asdownstream to the TT fragment. The preferred mode of realization of theinvention is upstream and is described in detail; a downstreamrealization is also contemplated. (Despite Halpern et al., J. Biol.Chem., (1993), 268(15):11188-11192, who indicated that thecarboxyl-terminal amino acids contain the domain required for binding topurified gangliosides and neuronal cells.)

[0066] The desired CNS area means, for example, the tongue which ischosen to direct the transport to hypoglossal motoneuron; the arm musclewhich is chosen to direct the transport to the spinal cord motoneurons.

[0067] For this realization of transplantation of a neuron to the CNS orthe spinal cord see Gage, F. H. et al. (1987), Neuroscience, 23:725-807,“Grafting genetically modified cells to the brain: possibilities for thefuture.”

[0068] The techniques for introducing the polynucleotides to cells aredescribed in U.S. Pat. Nos. 5,580,859 and 5,589,466, which is reliedupon and incorporated by reference herein. For example, the nucleotidesmay be introduced by transfection in vitro before reimplantation in areaof the CNS or the spinal cord.

[0069] A chemical linkage is considered for a particular embodiment ofthe invention and comprises the association between the TT fragment anda polynucleotide encoding the molecule of interest with its regulatoryelements, such as promoter and enhancer capable of expressing saidpolynucleotide. Then the TT fragment allows the retrograde axonaltransport and/or the transynaptic transport, and the product of thepolynucleotide is expressed directly in the neurons. This chemicallinkage can be covalent or not, but preferably covalent performed bythiolation reaction or by any other binding reaction as described in“Bioconjugate Techniques” from Gret T. Hermanson (Academic press, 1996).

[0070] The axonal retrograde transport begins at the muscle level, wherethe composition of interest is taken up at the neuromuscular junction,and migrates to the neuronal body of the motoneurons (which are alsocalled the first order neurons) in the CNS or spinal cord. First orderneurons mean neurons that have internalized the composition of interest,and thus in this case, correspond to motoneurons.

[0071] The transynaptic retrograde transport corresponds to interneuroncommunications via the synapses from the motoneurons, and comprisessecond order neurons and higher order neurons (fourth ordercorresponding to neurons in the cerebral cortex).

[0072] The different stages of the neuronal transport are through theneuromuscular junction, the motoneuron, also called first order neuron,the synapse at any stage between the neurons of different order, neuronof order second to fourth order, which corresponds to the cerebralcortex.

[0073] In one embodiment of this invention, it is shown that a β-gal-TTC(TT-fragment C) hybrid protein retains the biological activities of bothproteins in vivo. Therefore, the hybrid protein can undergo retrogradeand transneuronal transport through a chain of interconnected neurons,as traced by its enzymatic activity. These results are consistent withthose of others who used chemically conjugated TTC, or TTC fused toother proteins (12, 13, 14, 15). In these in vitro analyses, theactivity of the conjugated or hybrid proteins was likewise retained oronly weakly diminished. Depending on the nature of the TTC fusionpartner, different types of potential applications can be envisioned.For example, this application can be used to deliver a biologicallyactive protein into the CNS for therapeutic purposes. Such hybrid genescan also be used to analyze and map synaptically connected neurons ifreporters, such as lacZ or the green fluorescent protein (GFP; 29) gene,were fused to TTC.

[0074] The retrograde transport of the hybrid protein may bedemonstrated as follows. When injected into a muscle, β-gal activityrapidly localized to the somata of motoneurons that innervate themuscle. The arborization of the whole nerve, axon, somata and dendritescan easily be visualized. However, in comparison to the neurotropicviruses, the extent of retrograde transneuronal transport of the hybridprotein from the hypoglossal neurons indicates that only a subset ofinterconnected neurons is detected, although most areas containingsecond-order interneurons have been identified by the β-gal-TTC marker.Transneuronal uptake is mostly restricted to second order neurons. Insuch experiments, when a limited amount of a neuronal tracer is injectedinto a muscle or cell, only a fraction will be transported through asynapse, thereby imposing an experimental constraint on its detection.Presently, the most efficient method, in terms of the extent oftransport, relies on neurotropic viruses. Examples include: alpha-herpesviruses, such as herpes simplex type 1 (HSV-1), pseudorabies virus(PrV), and rhabdoviruses (24, 25). Viral methods are very sensitivebecause each time a virus infects a new cell, it replicates, therebyamplifying the signal and permitting visualization of higher orderneurons in a chain. Ultimately, however, one wants to map a neuronalnetwork in an in vivo situation such as a transgenic animal. Here, thedisadvantage of viral labeling is its potential toxicity. Most virusesare not innocuous for the neural cell, and their replication induces acellular response and sometimes cell degeneration (24). Furthermore,depending on experimental conditions, budding of the virus can occurleading to its spread into adjoining cells and tissues.

[0075] Differences in mechanisms of transneuronal migration could alsoaccount for the restricted number of neurons labeled by β-gal-TTC.Matteoli et al have provided strong evidence that the intact tetanustoxin crosses the synapses by parasitizing the physiological process ofsynaptic vesicle recycling at the nerve terminal (22). The toxinprobably binds to the inner surface of a synaptic vesicle during thetime the lumen is exposed to the external medium. Vesicle endocytosiswould then presumably provide the mechanism for internalization of thetoxin. Because the TTC fragment is known to mimic the migration of thetoxin in vivo, it could therefore direct the fusion protein along asimilar transynaptic pathway. If this hypothesis is confirmed, it wouldstrongly suggest that synaptic activity is required for thetransneuronal transport of β-gal-TTC. Therefore, only active neuronalcircuits would be detected by the hybrid protein. The possibledependence of β-gal-TTC on synaptic vesicle exocytosis and endocytosiscould be further investigated, since techniques are now available torecord synaptic activity in neural networks in vitro (30). In contrast,the transneuronal pathway of neurotropic viruses has not yet beenelucidated and could be fundamentally different, involving virus buddingin the vicinity of a synapse. Finally, the transneuronal transport ofthe hybrid protein might depend on a synaptic specificity, although thetetanus toxin is not known to display any (7, 23). It is thereforelikely that a virus would cross different or inactive synapses. Insummary, the restricted spectrum of interneuronal transport, in additionto its non-toxicity, make the β-gal-TTC hybrid protein a novel andpowerful tool for analysis of neural pathways.

[0076] One advantage of the fusion gene of the invention for neuronalmapping is that it derives from a single genetic entity that is amenableto genetic manipulation and engineering. Several years ago, a techniquebased on homologous recombination in embryonic stem cells was developedto specifically replace genes in the mouse (31, 32). This methodgenerates a null mutation in the substituted gene, although in aslightly modified strategy, a dicistronic messenger RNA can also beproduced (33, 34). When a reporter gene, such as E. coli lacZ, is usedas the substituting gene, this technique provides a means of marking themutated cells so that they can be followed during embryogenesis. Thus,this technique greatly simplifies the analysis of both the heterozygoteexpression of the targeted gene as well as the phenotype of null(homozygous) mutant animals.

[0077] Another advantage of this invention is that the compositioncomprising the fusion gene may encode an antigen or antigens. Thus, thecomposition may be used to elicit an immune response in its host andsubsequently confer protection of the host against the antigen orantigens expressed. These immunization methods are described in Robinsonet al., U.S. Pat. No. 5,43,578, which is herein incorporated byreference. In particular, the method of immunizing a patient or animalhost comprises introducing a DNA transcription unit encoding comprisingthe fusion gene of this invention, which encodes a desired antigen orantigens. The uptake of the DNA transcription unit by the host resultsin the expression of the desired antigen or antigens and the subsequentelicitation of humoral and/or cell-mediated immune responses.

[0078] Neural cells establish specific and complex networks ofinterconnected cells. If a gene were mutated in a given neural cell, wewould expect this mutation to have an impact on the functions of other,interconnected neural cells. With these considerations in mind, agenetic marker that can diffuse through active synapses would be veryuseful in analyzing the effect of the mutation. In heterozygous mutantanimals, the cells in which the targeted gene is normally transcribedcould be identified, as could the synaptically connected cells of aneural network. In a homozygous animal, the impact of the mutation onthe establishment or activity of the neural network could be determined.The feasibility of such an in vivo approach depends critically on theefficiency of synaptic transfer of the fusion protein, as well as itsstability and cellular localization.

[0079] Another extension of the invention is to gene therapy applied tothe CNS. This invention provides for uptake of a non-toxic,enzyme-vector conjugate by axon terminals and conveyance retrogradely tobrainstem motoneurons. A selective retrograde transynaptic mechanismsubsequently transports the hybrid protein into second-order connectedneurons. Such a pathway, which by-passes the blood-brain barrier, candeliver macromolecules to the CNS. In fact, pathogenic agents, such astetanus toxin and neurotropic viruses, are similarly taken up by nerveendings, internalized, and retrogradely transported to the nerve cellsomata. In such a scenario, the lacZ reporter would be replaced by agene encoding a protein that provides a necessary or interestingactivity and/or function. For example, the human CuZn superoxidedismutase, SOD-1, and the human enzyme β-N-acetylhexosaminidase A, HexA,have been fused or chemically coupled to the TTC fragment (13, 16), andtheir uptake by neurons in vitro was considerably increased and theirenzymatic functions partially conserved. Combined with the in vivoexperiments described here using β-gal-TTC, a gene therapy approachbased on TTC hybrid proteins appears to be a feasible method ofdelivering a biological function to the CNS. However, ways have to befound to target the TTC hybrid proteins, which are likely to besequestrated into vesicles, to the appropriate subcellular compartment.Such a therapeutic strategy could be particularly useful for treatingneurodegenerative and motoneuron diseases, such as amyotrophy lateralsclerosis (ALS, 35), spinal muscular atrophies (SMA, 36, 37), orneurodegenerative lysosomal storage diseases (38, 39). Injection intoselected muscles, even in utero, could help to specifically target theappropriate neurons. In addition, such an approach would avoid thesecondary and potentially toxic effects associated with the use ofdefective viruses to deliver a gene (40, 41).

EXAMPLE 1 Plasmid Constructions

[0080] (A) TTC Cloning:

[0081] Full length TTC DNA was generated from the genomic DNA from theClostridium Tetani strain (a gift from Dr. M. Popoff, Institut Pasteur)using PCR. Three overlapping fragments were synthesized: PCR1 of 465 bp(primer 1: 5′-CCC CCC GGG CCA CCA TGG TTT TTT CAA CAC CAA TTC CAT TTTCTT ATT C-3′ (SEQ ID NO:4) and primer 2: 5′-CTA AAC CAG TAA TTT CTG-3′(SEQ ID NO:5)), PCR2 of 648 bp (primer 3: 5′-AAT TAT GGA CTT TAA AAG ATTCCG C-3′ (SEQ ID NO:6) and primer 4: 5′-GGC ATT ATA ACC TAC TCT TAGAAT-3′ (SEQ ID NO:7)) and PCR3 of 338 bp (primer 5: 5′-AAT GCC TTT AATAAT CTT GAT AGA AAT-3′ (SEQ ID NO:8) and primer 6: 5′-CCC CCC GGG CATATG TCA TGA ACA TAT CAA TCT GTT TAA TC-3′ (SEQ ID NO:9)). The threefragments were sequentially introduced into pBluescript KS+ (Stratagene)to give pBS:TTC plasmid. The upstream primer 1 also contains anoptimized eukaryotic Ribosome Binding Site (RBS) and translationalinitiation signals. Our TTC fragment (462 amino acids) represents theamino acids 854-1315 of tetanus holotoxin, i.e. the carboxy-terminal 451amino acids of the heavy chain, which constitute the fragment C plus 11amino acids of the heavy chain that immediately precede the aminoterminus of the fragment C. The DNA sequence and amino acid sequence ofthe TTC fragment cloned in pBS:TTC is shown in FIG. 1. The constructpBS:TTC is shown in FIG. 2.

[0082] (B) pGEX:lacZ-TTC:

[0083] pGEX:lacZ was obtained by cloning a SmaI/XhoI lacZ fragment fromthe pGNA vector (a gift from Dr. H. Le Mouellic) into pGEX 4T-2(Pharmacia). PCR was used to convert the lacZ stop codon into an NcoIrestriction site. Two primers (upstream: 5′-CTG AAT ATC GAC GGT TTC CATATG-3′ (SEQ ID NO:10) and downstream: 5′-GGC AGT CTC GAG TCT AGA CCA TGGCTT TTT GAC ACC AGA C-3′ (SEQ ID NO:11)) were used to amplify thesequence between NdeI and XhoI, generating pGEX:lacZ(NcoI) frompGEX:lacZ. pGEX:lacZ-TTC was obtained by insertion of the TTC NcoI/XhoIfragment into pGEX:lacZ(NcoI), fusing TTC immediately downstream of thelacZ coding region and in the same reading frame. FIG. 3 shows thedetails of the pGEX:lacZ-TTC construct.

[0084] (C) pGEX:TTC-lacZ:

[0085] pBS:TTC was modified to change NcoI into a BamHI restriction site(linker 5′-CAT GAC TGG GGA TCC CCA GT-3′ (SEQ ID NO:12)) at the start ofthe TTC DNA, to give pBS:TTC(BamHI) plasmid. pGEX:TTC was obtained bycloning The TTC BamHI/SmaI fragment from pBS:TTC(BamHI) into pGEX 4T-2(Pharmacia). PCR was used to convert the TTC stop codon into an NheIrestriction site. Two primers (upstream: 5′-TAT GAT AAA AAT GCA TCT TTAGGA-3′ (SEQ ID NO:13) and downstream: 5′-TGG AGT CGA CGC TAG CAG GAT CATTTG TCC ATC CTT C-3′ (SEQ ID NO:14)) were used to amplify the sequencebetween NsiI and SmaI, generating pGEX:TTC(NheI) from pGEX:TTC. The lacZcDNA from plasmid pGNA was modified in its 5′ extremity to change SacIIinto an NheI restriction site (linker 5′-GCT AGC GC-3′ (SEQ ID NO:15)).pGEX:TTC-lacZ was obtained by insertion of the lacZ NheI/XhoI fragmentinto pGEX:TTC(NheI), fusing lacZ immediately downstream of the TTCcoding region and in the same reading frame. The details of theconstruct of pGEX:TTC-lacZ are shown in FIG. 4.

[0086] (D) pCMV:lacZ-TTC:

[0087] pCMV vector was obtained from pGFP-C1 (Clontech laboratories)after some modifications: GFP sequence was deleted by a BglII/NheIdigestion and relegation, and SacII in the polylinker was converted intoan AscI restriction site (linkers 5′-GAT ATC GGC GCG CCA GC-3′ (SEQ IDNO:16) and 5′-TGG CGC GCC GAT ATC GC-3′ (SEQ ID NO:17)).

[0088] pBluescript KS+ (Stratagene) was modified to change XhoI into anAscI restriction site (linker 5′-TCG ATG GCG CGC CA-3′ (SEQ ID NO:18)),giving pBS(AscI) plasmid. pBS:lacZ-TTC was obtained by cloning a XmaIlacZ-TTC fragment from pGEX:lacZ-TTC into pBS(AscI). pCMV:lacZ-TTC wasobtained by insertion of the lacZ-TTC XmnI/AscI fragment into pCMVvector at the XhoI and AscI sites (XhoI and XmnI was eliminated with theclonage), putting the fusion downstream of the CMV promotor. FIG. 8shows the details of the construct pCMV:lacZ-TTC. Plasmid pCMV:lacZ-TTCwas deposited on Aug. 12, 1997, at the Collection Nationale de Culturesde Microorganisms (CNCM), Institut Pasteur, 25, Rue de Docteur Roux,F-75724, Paris Cedex 15, France, under Accession No. I-1912.

EXAMPLE 2 Purification of the Hybrid Protein

[0089] The E. coli strain SR3315 (a gift from Dr. A. Pugsley, InstitutPasteur) transfected with pGEX:lacz-TTC was used for protein production.An overnight bacterial culture was diluted 1:100 in LB medium containing100 μg/ml ampicillin, and grown for several hours at 32° C. until an ODof 0.5 was reached. Induction from the Ptac promoter was achieved by theaddition of 1 mM IPTG and 1 mM MgCl₂ and a further 2 hrs incubation. Theinduced bacteria were pelleted by centrifugation for 20 min at 3000 rpm,washed with PBS and resuspended in lysis buffer containing 0.1 M Tris pH7.8, 0.1M NaCl, 20% glycerol, 10 mM EDTA, 0.1% Triton-X100, 4 mM DTT, 1mg/ml lysosyme, and a mixture of anti-proteases (100 μg/ml Pefablok, 1μg/ml leupeptin, 1 μg/ml pepstatin, 1 mM benzamidine). After celldisruption in a French Press, total bacterial lysate was centrifuged for10 min at 30000 rpm. The resulting supernatant was incubated overnightat 4° C. with the affinity matrix Glutathione Sepharose 4B (Stratagene)with slow agitation. After centrifugation for 5 min at 3000 rpm, thematrix was washed three times with the same lysis buffer but withoutlysosyme and glycerol, and then three times with PBS. The resin wasincubated overnight at 4° C. with Thrombin (10 U/ml; Sigma) in PBS inorder to cleave the β-gal-TTC fusion protein from theGlutatione-S-transferase (GST) sequence and thereby elute it from theaffinity column. Concentration of the eluted fusion protein was achievedby centrifugation in centricon X-100 tubes (Amicon; 100,000 MW cutoffmembrane).

[0090] Purified hybrid protein was analyzed by Western blotting afterelectrophoretic separation in 8% acrylamide SDS/PAGE under reducingconditions followed by electrophoretic transfer onto nitrocellulosemembranes (0.2 mm porosity, BioRad). Immunodetection of blotted proteinswas performed with a Vectastaln ABC-alkaline phosphatase kit (VectorLaboratories) and DAB color development. Antibodies were used asfollows: rabbit anti-β-gal antisera (Capel), dilution 1:1000; rabbitanti-TTC antisera (Calbiochem), dilution 1:20000. A major band with arelative molecular mass of 180 kDa corresponding to the β-Gal-TTC hybridprotein was detected with both anti-β-Gal anti-TTC antibodies.

EXAMPLE 3 Binding and Internalization of Recombinant Protein inDifferentiated 1009 Cells

[0091] The 1009 cell line was derived from a spontaneous testicularteratocarcinoma arising in a recombinant inbred mouse strain (129×B6)(17). The 1009 cells were grown in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal calf serum and passaged at subconfluence. Invitro differentiation with retinoic acid and cAMP was performed asdescribed (18). Eight days after retinoic acid treatment, cells wereused for the internalization experiments with either the hybrid proteinor β-gal.

[0092] Binding and internalization of the β-Gal-TTC fusion were assessedusing a modified protocol (16). Differentiated 1009 cells were incubatedfor 2 hrs at 37° C. with 5 μg/ml of p-Gal-TTC or p-Gal protein dilutedin binding buffer (0.25% sucrose, 20 mM Tris acetate 1 mM CaCl2, 1 mMMgCl₂, 0.25% bovine serum albumin, in PBS). The cells were thenincubated with 1 μg/ml Pronase E (Sigma) in PBS for 10 min at 37° C.,followed by washing with proteases inhibitors diluted in PBS (100 μg/mlPefablok, 1 mM benzamidine).

[0093] The cells were fixed with 4% formalin in PBS for 10 min at roomtemperature (RT) and then washed extensively with PBS. β-gal activitywas detected on fixed cells by an overnight staining at 37° C. in X-Galsolution (0.8 mg/ml X-Gal, 4 mM potassium ferricyanide, 4 mM potassiumferrocyanide, 4 mM MgCl₂ in PBS). For electron microscopy, the cellswere further fixed in 2.5% glutaraldehyde for 18 hrs, and then processedas described (19).

[0094] For immunohistochemical labeling, cells were fixed with 4%paraformaldehyde in PBS for 10 min at RT then washed extensively withPBS, followed by a 1 hr incubation at RT with 2% BSA/0.02% Triton X-100in PBS. Cells were co-incubated in primary antibodies diluted in 2%BSA/0.02% Triton X-100 in PBS for 2 hrs at RT. Antibodies used were amouse anti-neurofilament antibody (NF 200 Kd, dilution 1:50; Sigma) orthe rabbit anti-TTC antibody (dilution 1:1000). The labeling wasvisualized using fluorescent secondary antibodies: Cy3, goat anti-rabbitIgG (dilution 1:500; Amersham) or anti-mouse IgG with extravidin-FITC(dilution 1:200; Sigma). Cells were mounted in moviol and visualizedwith epifluorescence.

EXAMPLE 4 In Vivo Recombinant Protein Injection

[0095] 14-week old B6D2 μl mice were obtained from IFFA-CREDO. Theanimal's tongue muscle was injected using an Hamilton syringe (20 μl peranimal) while under general anesthesia with 3% Avertin (15 μl/g ofanimal). The protein concentration was 0.5 to 5 μg/μl in PBS; therefore,mice received approximately 10 to 100 μg per injection. Animals werekept alive for 12 hrs to 48 hrs post-injection to permit migration ofthe injected protein, and in no case were any tetanus symptoms detected.The mice were sacrificed by intracardiac perfusion with 4%paraformaldehyde in PBS while under deep anesthesia. Brains wereharvested, rinsed in PBS and incubated in 15% sucrose overnight at 4°C., then mounted in tissue-tek before sectioning, 15 μm thick slicesusing a cryostat.

EXAMPLE 5 Histology, Immunohistology, and X-Gal Staining

[0096] For in toto X-Gal staining of the dissected brain and tongue,mice (10 animals) were sacrificed and fixed as described above. Thebrain was further cut with a scalpel along a median plane and directlyincubated for 12 hrs in X-Gal solution.

[0097] For immunohistology, sections were incubated In a 1:5000 dilutionof anti-TTC antibody in 2% BSA/0.02% Triton X-100 in PBS overnight at 4°C. after nonspecific antibody binding sites were blocked by a 1 hrincubation in the same buffer. Antibody detection was carried out usingthe Vectastain ABC-alkaline phosphatase kit with DAB color development.For X-Gal staining, sections were incubated in X-Gal solution andcounterstained for 30 sec with hematoxylin 115 (v/v) in PBS. Histologyon adjacent sections was done after X-Gal staining, using a 30 secincubation in hematoxylin/thionin solution. All sections were mounted inmoviol before eight microscopy analysis.

EXAMPLE 6A Internalization of the β-gal-TTC Fusion Protein by Neurons InVitro

[0098] Differentiation of 1009 cells with retinoic acid and cAMP invitro yields neuronal and glial cells (18, 20). X-Gal staining orimmunolabeling were performed after incubation with the β-gal-TTC fusionprotein or with either the β-gal or TTC proteins alone. Only when thehybrid protein was incubated with differentiated 1009 cells was a strongX-Gal staining detected in cells having a neuronal phenotype. No signalwas detected when β-gal alone was incubated under the same conditions. Asimilar X-Gal staining pattern was obtained after pronase treatment ofthe cells to remove surface bound proteins, indicating that the hybridprotein had been internalized. The intracellular localization of thehybrid protein was further confirmed by electron microscopic analysis ofX-Gal-stained cells. Furthermore, the enzymatic activity observed inaxons seemed to be localized in vesicles associated with filaments,which is in agreement with previous work on TTC fragment or nativetetanus toxin (14, 21, 22). Co-labeling with anti-TTC andanti-neurofilament antibodies revealed that β-gal activity co-localizedwith TTC fragment in neuronal cells. No glial cells were labeled witheither antibody.

EXAMPLE 6B Internalization of the TTC-β-gal Fusion Protein by Neurons InVitro

[0099] The method used for the internalization was identical to thatdescribed in Example 6 above. The results show efficientlyinternalization of the hybrid as in Example 6 above.

EXAMPLE 7 Retrograde Transport of the Hybrid Protein In Vivo

[0100] To study the behavior of the β-gal-TTC protein in vivo, thehybrid protein was tested in a well characterized neuronal network, thehypoglossal system. After intramuscular injection of β-gal-TTC proteininto the mouse tongue, the distribution of the hybrid protein in the CNSwas analyzed by X-Gal staining. Various dilutions of the protein wereinjected and sequential time points were analyzed to permit proteintransport into hypoglossal motoneurons (XII), and its furthertransneuronal migration into connected second order neurons.

[0101] A well-defined profile of large, apparently retrogradely labeledneurons was clearly evident in the hypoglossal structure, analyzed intoto at 12 hrs post-injection. A strong labeling was also apparent inthe hypoglossal nerve (XIIn) of the tongue of the injected mice. At thelevel of muscle fibers, button structures were observed that mightreflect labeling of neuromuscular junctions where the hybrid protein wasinternalized into nerve axons. These data demonstrate that the β-gal-TTChybrid protein can migrate rapidly by retrograde axonal transport as faras motoneuron cell bodies, after prior uptake by nerve terminals in thetongue. This specific uptake and the intraaxonal transport are similarto the properties that have been described for the native toxin (6, 21,23).

[0102] Transport of the hybrid protein was examined in greater detail byanalyzing X-Gal-stained brain sections. Motoneurons of the hypoglossalnucleus became labeled rapidly, with 12 hrs being the earliest timepoint examined. Most of the label was confined to neuronal somata, thecell nuclei being unlabeled. The intensity of the labeling depends uponthe concentration of the β-gal-TTC protein injected: when 10 μg ofprotein was injected, only the hypoglossal somata were detected, whereaswith 25 to 50 μg a fuzzy network of dendrites was visualized;transynaptic transfer was detected with 100 μg of hybrid protein. Anidentical distribution of label was observed then brain sections wereimmunostained with an anti-TTC antibody, demonstrating that β-gal andTTC fragment co-localize within cells. Finally, injection of β-gal alonedid not result in labeling of the hypoglossal nuclei and thereforeconfirms that transport of the hybrid protein is TTC-dependent. Labelingwith an anti-TTC antibody was less informative than detection of β-galactivity; for instance, the nerve pathway to the brain could not bevisualized by anti-TTC immunostaining. At 18 hrs post-injection,labeling was observed in the hypoglossal nuclei: all motoneuron cellbodies and the most proximal part of their dendrites were very denselystained. In contrast, no labeling was ever detected in glial cellsadjoining XII motoneurons or their axons. Our results are in accordancewith others who reported an identical pattern of immunolabeling afterinjection of the TTC fragment alone (9). Transneuronal transfer isdetectable after 24 hrs. An additional 24 hrs and beyond did not yield adifferent staining.

EXAMPLE 8 Transneuronal Transport of the Hybrid Protein

[0103] Second order interneurons, as well as higher order neurons thatsynapse with the hypoglossal motoneurons, have been extensively analyzedusing conventional markers, such as the wheat germagglutinin-horseradish peroxidase complex (WGA-HRP) or neurotropicviruses such as alpha-herpes (24) and rhabdoviruses (25). An exhaustivecompilation of regions in the brain that synaptically connect to thehypoglossal nucleus has also been described recently (25). In thisinvention, the distribution of the β-gal-TTC fusion depended on theinitial concentration of protein injected into the muscle and the timeallowed for transport after injection. Up to 24 hrs post-injection,labeling was restricted to the hypoglossal nuclei. After 24 hrs, thedistribution of second order transneuronally labeled cells in variousregions of the brain was consistent and reproducible. Even at longertime points (e.g. 48 hrs), labeling of the hypoglossal nucleus remainedconstant. At higher magnification, a discrete and localized staining ofsecond-order neurons was observed, suggesting that the hybrid proteinhad been targeted to vesicles within cell somata, synapses and axons. Asimilar patchy distribution was previously described for tetanus toxinand TTC fragment alone (14, 21, 22).

[0104] Intense transneuronal labeling was detected in the lateralreticular formation (LRF), where medullary reticular neurons have beenreported to form numerous projections onto the hypoglossal nucleus (26,27). β-gal activity was detected bilaterally in these sections. Labelled LRF projections formed a continuous column along the rostrocaudalaxis, beginning lateral to the hypoglossal nucleus, with a few neuronsbeing preferentially stained in the medullary reticular dorsal (MdD) andthe medullary reticular ventral (MdV) nuclei. This column extendsrostrally through the medulla, with neurons more intensely labeled inthe parvicellular reticular nucleus (PCRt, caudal and rostral). After 48hrs, cells in MdD and PCRt were more intensely stained. A secondbilateral distribution of medullary neurons projecting to thehypoglossal nucleus was detected in the solitary nucleus (Sol) but thelabeling was less intense than in the reticular formation, presumablybecause relatively few cells of the solitary nucleus project onto thehypoglossal nucleus (26). However, no labeling was found in the spinaltrigeminal nucleus (Sp5), which has also been shown to project onto thehypoglossal nucleus (26). Transynaptic transport of the β-gal-TTCprotein was also detected in the pontine reticular nucleus caudal (PnC),the locus coeruleus (LC), the medial vestibular nucleus (MVe) and in afew cells of the inferior vestibular nucleus (IV). These cell groups areknown to project onto the hypoglossal nucleus (25), but their labelingwas weak, probably because of the greater length of their axons. A fewlabeled cells were observed in the dorsal paragigantocellular nucleus(DPGi), the magnocellular nucleus caudal (RMc), and the caudal raphenucleus (R); their connections to the hypoglossal nucleus have also beenreported (25). Finally, labeled neurons were detected bilaterally inmidbrain projections, such as those of the mesencephalic trigeminalnucleus (Me5), and a few neurons were stained in the mesencephaliccentral gray region (CG). These latter nuclei have been typed asputative third order cell groups related to the hypoglossal nucleus(25).

[0105] Neurons in the motor trigeminal nucleus (Mo5) and the accessorytrigeminal tract (Acs5) were also labeled, along with a population ofneurons in the facial nucleus (N7). However, interpretation of thislabeling is more ambiguous, since it is known that motoneurons in thesenuclei also innervate other parts of the muscular tissue, and diffusionof the hybrid protein might have occurred at the point of injection.Conversely, these nuclei may have also projected to the tonguemusculature via nerve XII, since neurons in N7 have been reported toreceive direct hypoglossal nerve input (28). This latter explanation isconsistent with the fact that labeling in these nuclei was detected onlyafter 24 hrs; however, this point was not further investigated.

[0106] Together, the data summarized in Table 1 clearly establishtransneuronal transport of the β-gal-TTC fusion protein from thehypoglossal neurons into several connected regions of the brainstem.TABLE 1 Transneuronal transport of the lacZ-TTC fusion from the XIInerve: labeling of different cells types in the central nervous system.Cell groups 12-18 hrs 24-48 hrs First order neurons First category: XII,hypoglossal motoneurons ++ +++ Second category: N7, facial nu − ++ Mo5,motor trigeminal nu − ++ Acs5, accessory trigeminal nu − + Second ordercell groups MdD, medullary reticular nu, dorsal − ++ MdV, medullaryreticular nu, ventral − +/− PCRt, parvicellular reticular nu, caudal −++ PCRt, parvicellular reticular nu, rostral − ++ Sol, solitary tract nu− + DPGi, dorsal paragigantocellular nu − +/− PnC, pontine reticular nu,caudal − + RMc, magnocellular reticular nu − +/− R, caudal raphe nu −+/− MVe, medial vestibular nu − + IV, inferior vestibular nu − +/− LC,locus coeruleus − + Me5, mesencephalic trigeminal nu (*) − + CG,mesenphalic central gray (*) − +/−

[0107] In another embodiment of the invention, we have constructed afusion protein (GFP-TTC) comprising the C-terminal fragment of tetanustoxin and the GFP reporter gene, and have demonstrated its effectivenessto map a simple neural network retrogradely and transsynaptically intransgenic mice. (Maskos et al., 2002). The GFP-TTC fusion proteinpermits the visualization of membrane traffic at the presynaptic levelof the neuromuscular junction and can be detected opticaly withoutimmunological or enzymatic reactions. The GFP-TTC fusion protein,therefore, permits observation of active neurons with minimaldisturbance of their physiological activities.

[0108] We have also previously shown that, without neural activity,localization of a TTC fusion protein at the NMJ is impaired (Miana-Menaet al., 2002). In this aspect of the invention, therefore, weinvestigated in vivo, the influence of neurotrophic factors on neuronallocalization and internalization of GFP-TTC and the mechanisms by whichcertain neurotrophic factors influence neuronal trafficking in vivo. Wefound that localization of GFP-TTC at the NMJ is rapidly induced byneurotrophic factors such as Brain Derivated Neurotrophic Factor (BDNF),Neurotrophin 4 (NT-4), and Glial-Derived Neurotrophic Factor (GDNF) butnot by Nerve Growth Factor (NGF), Neurotrophin 3 (NT-3), and CiliaryNeurotrophic Factor (CNTF).

[0109] Co-injection of various amounts of BDNF with the GFP-TTC probeinduces an increase of the fluorescence measured at the neuromuscularjunction (NMJ). This effect, which is detectable as early as 5 min afterinjection and reaches a maximum level at about 30 min after injection,indicates that BDNF treatment enhances neuronal endocytosis. Among otherfunctions, BDNF stimulates the secretion of neurotransmitter fromXenopus nerve muscle co-cultures and from hippocampal neurons (Lohof etal., 1993; Tyler and Pozzo-Miller, 2001). Since tetanus toxin is knownto enter neurons by means of synaptic vesicle endocytosis (Matteoli etal., 1996), BDNF might increase GFP-TTC internalization throughenhancement of synaptic vesicle turnover. In our study, BDNF effectspersisted after BoTx/A treatment, which blocks exocytosis andendocytosis of synaptic vesicles, showing that BDNF increases thekinetics and localization of a TTC-containing fusion protein at the NMJthrough another endocytic pathway. Therefore, intramuscular injection ofGFP-TTC and visualization of transport mechanisms revealed at least twodifferent endocytic pathways: a clathrin-dependent and aclathrin-independant pathways. We found that after intramuscularinjection of GFP-TTC, it displayed characteristics consistent withlocalization in lipid rafts, including biochemical colocalization withcaveolin 3 and colocalization with GM1, a raft marker identified by CT-bbinding (Orlandi and Fishman, 1998; Wolf et al., 1998). Accordingly, theclathrin-independent pathway used by GFP-TTC, appears to involve lipidmicrodomains. Analysis by confocal microscopy revealed morphologicallytwo different labelings. Firstly, a GFP-TTC diffuse staining, whichpartially overlaps with the synaptic vesicle SV2 but also with the raftmarker CT-b, indicating a mixing of synaptic vesicles and lipid rafts.Secondly, highly fluorescent domains, which are detected before andpersist after the more diffuse pattern and that appear to beinvaginations or infoldings of the synaptic membrane. These GFP-TTCpatches contained only CT-b labeling. Indeed, lipid microdomains whichplay a role in cellular functions such as vesicular trafficking andsignal transduction (Simons and Toomre, 2000), can move laterally andcluster into larger patches (Harder et al., 1998). They might also bespecific zones of exocytosis in the presynaptic compartment, undergoinga rapid form of internal traffic in response to retrograde signalingfrom target cells. Similar infolding and cisternae structures have beendescribed in frog motor nerve terminals which replesnish the pool ofsynaptic vesicles in a manner dependent upon neuronal activity (Richardset al., 2000). In CHO cells, tubular caveolae have also been described(Mundy et al., 2002).

[0110] Based on the kinetics of probes for NMJ localization, we observeddifferent trafficking behaviours for GFP-TTC and CT-b. It has beenpostulated that targeting of toxin into the cell depends on thestructure and function of endogenous ganglioside receptors, which couldcouple toxins to specific lipid raft microdomains (Wolf et al., 1998).Thus, in vivo, endogenous or injected BDNF might increase the amount oflipid microdomains containing TTC receptors. Tetanus toxin and choleratoxin bind to different gangliosides, known as GD1b/GT1b and GM1,respectively. Hence, the difference we observed in the dynamics ofrecruitment at the presynaptic motor nerve terminal may be relevant todifferent lipid microdomains having specific glycosphingolipids andprotein composition. Neuronal membranes are rich in gangliosides anddifferent microdomains are likely to co-exist on the cell surface.Indeed, Thy-1 and PrP prion protein, two functionally different GPIproteins, are found in adjacent microdomains (Madore et al., 1999).Similarly, syntaxins are concentrated in cholesterol-dependentmicrodomains, which are distinct from rafts containing GPI-linkedproteins (Lang et al., 2001).

[0111] Like BDNF, NT-4 was also found to increase the concentration ofGFP-TTC at the NMJ, whereas NGF and NT-3 had no effect. Since the TrkBreceptor is specifically activated by BDNF and NT-4, TrkB activationmight be involved in this neoronal trafficking. Interestingly,high-frequency neuronal activity and synaptic transmission have beenshown to elevate the number of TrkB receptors on the surface of culturedhippocampal neurons (Du et al., 2000), apparently by recruiting extraTrkB receptors to the plasma membrane (Meyer-Franke et al., 1998).Moreover, TrkB is highly enriched in lipid microdomains from neuronalplasma membrane (Wu et al., 1997). However, no specific colocalizationbetween GFP-TTC and TrkB or p-Trk receptors were detected at the NMJ.Thus, TrkB may act indirectly on the detected traffic at the presynapticmotor nerve membrane.

[0112] It is worth noting that the TTC fragment has been detected incultured motoneurons in the same vesicles as p75^(NTR) (Lalli andSchiavo, 2002). This colocalization may be explained by the tightassociation of p75^(NTR), which is expressed mainly during developmentand in pathological conditions, with GT1b ganglioside (Yamashita et al.,2002). Binding of neurotrophins to their Trk receptors leads tophosphorylation of tyrosine residues that are recognized by severalintracellular signaling proteins. Such interactions lead to theactivation, by means of a kinase cascade, of the MAP kinase, PI 3-kinaseand phospholipase-C-γ pathways (for review see (Huang and Reichardt,2003)). Many of the intermediates in these signaling cascades are alsopresent in lipid rafts (Simons and Toomre, 2000; Tsui-Pierchala et al.,2002). Activation of PKA is required for translocation of activatedp75^(NTR) to lipid rafts (Higuchi et al., 2003). Similarly, thecoreceptor GFRα1, which binds GDNF and thus allows activation of thec-RET tyrosine kinase receptor, localize to lipid rafts. GFRα1 recruitsRET to lipid rafts after GDNF stimulation and results in strong andcontinuous signal transduction (Paratcha et al., 2001; Tansey et al.,2000).

[0113] Another neurotrophic factor, GDNF, also induced GFP-TTClocalization at the NMJ. GDNF, however, activates a different receptor(i.e., a GFRα/cRET receptor) than BDNF and NT-4. Because BDNF/NT-4 andGDNF activate different receptors, we postulated that component(s) oftheir activation pathways may activate the trafficking of GFP-TTCreceptors in specific lipid microdomains. Indeed, various stimuli canlead to internalization of caveolae, a specialized form of lipid rafts.Thus, simian virus 40 stimulates its internalization in caveolae andtransport via caveosomes (Pelkmans et al., 2001). Similarly, thealbumin-docking protein pg60 activates its transendothelial transport byinteraction with caveolin-1 and subsequent activation of Src kinasesignaling (Minshall et al., 2000). Recently, it has been reported thattetanus toxin can activate, through the TTC fragment, intracellularpathways involving Trk receptors, extracellular signal-regulated kinases(ERK) and protein kinase C isoforms (Gil et al., 2001; Gil et al., 2000;Gil et al., 2003). In this way, tetanus toxin could thereforeautoactivate its neuronal endocytosis via an uncoated pathway ratherthan by clathrin-dependent pathway to avoid the lysosomal degradation.

[0114] Finally, we have demonstrated that GFP-TTC trafficking isregulated by neurotrophic factors. By visualization of GFP-TTCtrafficking, our data show that BDNF can stimulate both clathrin-coatedand uncoated endocytic pathways, presumably via TrkB activation. Sincetetanus toxin, as other pathogens or toxins, uses constitutivemechanisms for its internalization and traffic in cells, we have beenable to visualize with GFP-TTC, a physiological response to neurotrophicfactors.

[0115] This aspect of the invention is further discussed in thefollowing examples.

EXAMPLE 9 GFP-TTC Localization at the NMJ

[0116] To determine the characteristics of the GFP-TTC distribution atthe NMJ, a single injection of the GFP-TTC fusion protein was performedin the immediate vicinity of the Levator auris longus (LAL) muscle andat various times after the injection, the LAL was removed and examinedas a whole mount. As LAL is a thin and flat muscle consisting of only afew layers of fibers, the entirety of the neuromuscular preparation withassociated nerves could be examined by confocal analysis (FIG. 6A). Asshown in FIG. 6, GFP-TTC rapidly concentrates at the NMJ, as identifiedby the staining of muscle nicotinic acetylcholine receptors withTRITC-conjugated α-bungarotoxine (α-BTX). A patchy clustering of GFP-TTCwas observed after approximately 5 min following the deposit of thefusion protein onto the surface of the LAL muscle (FIGS. 6D and D′).After 30 min, a more diffuse staining was observed that was distributedover the entire surface of the NMJ (FIGS. 6E and E′), and whichpersisted for about 2 h (FIGS. 6F and F′). Immunostaining experiments,performed with an antibody that recognizes troponin T confirmed thatGFP-TTC is concentrated mainly in presynaptic motor nerve terminals ofthe NMJ (FIGS. 6C and C′). We could also detect a strong GFP-TTClabeling at the nodes of Ranvier of intramuscular myelinated axons andin sensory nerve fibers (FIGS. 6A and B; arrows and arrowheadsrespectively). It is likely that most of the GFP-TTC probe wasinternalized within 24 h, since only a few fluorescent patches persistedat the NMJ 24 h after its injection (FIGS. 6G and G′).

EXAMPLE 10 Influence of BDNF on GFP-TTC Trafficking in Motor NerveTerminals

[0117] To assess whether exogenously applied neurotrophins affectedGFP-TTC recruitment in motor nerve terminals, increasing concentrationsof BDNF (2.5-250 ng) were co-injected with GFP-TTC in the vicinity ofLAL muscles, while control mice were injected with GFP-TTC alone. Micewere sacrificied and LAL muscles harvested 30 min after injection. GFPfluorescence was quantified by confocal microscopy analysis at NMJs,after identification by TRITC-A-BTX labeling. BDNF injection produced astatistically significant concentration-dependent enhancement of GFP-TTCfluorescence at the NMJ, with the highest effect obtained with 50 ngBDNF (FIG. 7B and Table 2) while higher doses (100 and 250 ng) resultedin weaker elevations in the level of GFP-TTC concentration at the NMJ(1.72±0.12 and 1.15±0.22 fold respectively). The higher GFP-TTC axonallabeling observed at these higher doses (FIG. 7A, arrows), probablycorrelates to an enhanced internalization of the probe.

[0118] In TrkB mutant mice, a physiological phenotype in the facialnerve nucleus, which innervates LAL muscle has been reported (Klein etal., 1993; Silas-Santiago et al., 1997). To exclude the possibility thatthe BDNF effect observed could be LAL specific, a different muscle, thegastrocnemius, was also analyzed. Thirty minutes after injecting GFP-TTC(+BDNF 50 ng) in gastrocnemius, muscles were fixed, removed and seriallysectioned. For each muscle, different serial sections were quantifiedfor GFP-TTC fluorescence at the motor nerve terminals as described inmaterial and methods. We found that the BDNF-dependent increase ofGFP-TTC concentration at the NMJ, closely resembled that observed in LAL(1.51±0.12 fold increase vs 2.12±0.19 respectively).

EXAMPLE 11 Influence of Other Neurotrophic Factors on GFP-TTCLocalization at Motor Nerve Terminals

[0119] We also examined the effect of five additional trophic factors onGFP-TTC localization at the NMJ, including the neurotrophins NT-3; NT-4and NGF as well as the neurocytokine CNTF (Ciliary Neurotrophic Factor),a member of the LIF cytokine family, and GDNF (Glial-DerivatedNeurotrophic Factor), a member of the TGF-β superfamily (Table 2). ManyBDNF actions in neurons are mediated via the high affinity receptortyrosine kinase TrkB, which is also the receptor for NT-4. Like BDNF,NT-4 also induced GFP-TTC localization at the NMJ (a 1.54±0.23 foldincrease). A level of induction similar to NT-4 was also observed forGDNF (Table 2). On the other hand, even at high concentrations, neitherNGF, NT-3, nor CNTF exhibited a significant effect on GFP-TTClocalization. TABLE 2 Effect of various neurotrophic factors on nerveterminal's GFP-fluorescence level 30 min after in vivo GFP-TTCinjection. Relative increase in Receptor fluorescence level BDNF TrkB2.12 ± 0.19** NT-4 TrkB 1.49 ± 0.23** NT-3 TrkC 0.94 ± 0.05 NGF TrkA1.06 ± 0.06 CNTF CNTFRα 0.95 ± 0.05 GDNF GFRα/cRET 1.51 ± 0.02*

EXAMPLE 12 Comparison of Trk Receptors Distribution and GFP-TTCLocalization at Motor Nerve Endings

[0120] Detection of either TrkB mRNA or protein in adult skeletal muscleand motoneurons has been reported in several studies (Funakoshi et al.,1993; Gonzalez et al., 1999; Griesbeck et al., 1995; Yan et al., 1997).Since our results indicated that the BDNF effect on GFP-TTC localizationis dependent on TrkB receptor activation, it was of interest todetermine whether GFP-TTC colocalized with TrkB at the NMJ of LALmuscles. Consistent with previous studies (Gonzalez et al., 1999; Sakumaet al., 2001), TrkB immunostaining was confined to the NMJ (FIG. 8). Inthe presynaptic side, TrkB staining was adjacent to, but not colocalizedto the clusters of GFP-TTC labeling. Similar results were also obtainedwith an antibody that recognizes the activated Trk receptors (p-Trk,data not shown). This observation suggests that the mechanism wherebyBDNF has an influence on the concentration of GFP-TTC at the nerveterminals, does not involve a direct interaction between TrkB andGFP-TTC or its receptors.

EXAMPLE 13 Mechanisms Involved in BDNF Effect on GFP-TTC Concentrationat the NMJ

[0121] Possible explanations for the BDNF-induced enrichment of GFP-TTCat the NMJ could involve an elevated rate of localization of the probeat the NMJ, and/or an increased neuronal endocytosis of the probe.Confocal analysis performed 5, 15, 30, 60 and 120 min after GFP-TTCinjection (+BDNF 50 ng) showed maximal labeling intensity at 30 min withBDNF injection, whereas in controls, the maximal staining occurred at 1h and reached a level lower than that obtained with BDNF co-injection.After the first hour, similar levels of GFP-TTC were recorded at the NMJin both conditions (FIG. 9A). These results are in accordance withprevious results in Xenopus nerve-muscle co-culture indicating atime-limiting effect of BDNF (Lohof et al., 1993).

[0122] In vitro, tetanus neurotoxin internalization in neurons appearsto involve both coated and uncoated-vesicular pathways (Herreros et al.,2001; Matteoli et al., 1996). Experiments performed either in vitro onexcised LAL muscles with the endocytic fluid marker RH414 (data notshown), or immunostained against the SV2 synaptic vesicle proteins (FIG.9B) and synaptophysin (data not shown) showed some overlapping withGFP-TTC labeling, indicating that the endocytosis of GFP-TTC was in partvia recycling of neuronal synaptic vesicles. To differentiate betweenclathrin-dependent and clathrin-independent endocytic pathways, we usedtreatment with botulinum neurotoxin serotype A (BoTx/A), which blocksneurotransmitter release and endocytosis in motor nerve terminals (dePaiva et al., 1999). When BoTx/A was applied 48 hours before GFP-TTCinjection, the probe level at the NMJ was markedly decreased by 50%(FIG. 9C), indicating that both clathrin-dependent and independentpathways are used to a comparable degree.

[0123] Enhanced synaptic transmission produced by application ofexogenous BDNF; NT-3 or NT-4 involves a potentiation of neurotransmitterrelease (Lohof et al., 1993; Stoop and Poo, 1996; Wang and Poo, 1997).The increasing amount of GFP-TTC at the NMJ induced by BDNF injectioncould therefore be due in part to an elevated recycling of synapticvesicles. To explore this hypothesis, increased exocytosis andendocytosis of synaptic vesicles were induced by GFP-TTC injection in ahigh-potassium medium. Five minutes after injection, exposure to high K⁺medium or BDNF induced a similar increase of GFP-TTC level at the NMJ.However, after 30 min, the effect of high K⁺ was no longer detectable,whereas maximal induction was reached with BNDF at this time (FIG. 9D).Finally, even after neurotransmitter release and synaptic vesiclerecycling were blocked by BoTx/A, an increased GFP-TTC signal wasinduced by BDNF treatment (FIG. 9C) with an amplitude comparable to thatrecorded in the non-paralyzed control NMJ (2.05 fold increase vs 2.12respectively). Taken together, these results indicate that BDNF enhancesan alternative endocytic pathway that appears to involve uncoatedvesicles.

EXAMPLE 14 Evidence for Association of GFP-TTC in Detergent-InsolubleMembrane at the Neuromuscular Junction

[0124] Binding of TTC to plasma membranes involves association topolysialogangliosides GD1b and GT1b, as well as a N-glycosylated 15 kDaprotein. These three components partition preferentially in membranemicrodomains called rafts. In vitro, TTC has been shown to associatewith such microdomains in NGF-differentiated PC12 cells and in culturedspinal cord neurons (Herreros et al., 2001; Vyas, 2001). To test in vivowhether GFP-TTC associated to lipid rafts, gastrocnemius muscles weresubmitted to detergent extraction to isolate lipid microdomains afterGFP-TTC intramuscular injection. Twelve fractions from the discontinuoussucrose gradient were collected and analyzed for distribution ofGFP-TTC.

[0125] Neurons do not contain caveolin or morphologically distinctcaveolae (Anderson, 1998), but significant fractions of cholesterol andglycosphingolipids are found in detergent-insoluble complexes, which areindistinguishable using the criteria of detergent insolubility fromthose associated with caveolae (Schnitzer et al., 1995). Thus, caveolin3, a specific muscular caveolar marker (Tang et al., 1996), was used toidentify the detergent-resistant fractions. Immunoblot analysis revealedthat GFP-TTC co-migrated with raft microdomains, which contain caveolin3 (FIG. 10A).

[0126] To investigate whether the GFP-TTC patches observed in vivo inmotor nerve terminals correspond to lipid microdomains, we performedco-staining with Alexa 594-conjugated cholera toxin-B fragment (CT-b).CT-b specifically binds to ganglioside GM1, which is enriched incholesterol-rich membrane microdomains, and is commonly used as a markerfor membrane rafts and caveolae (Orlandi and Fishman, 1998; Schnitzer etal., 1995; Wolf et al., 1998). GFP-TTC and Alexa 594-conjugated CT-bfragment were co-injected into the gastrocnemius and confocal analysiswas performed 1; 3; 5; 9 and 24 h later, with the NMJ being identifiedby AlexaFluor 647-conjugated α-BTX (FIG. 10B). Although the GFP-TTClabeling of motor nerve terminals was easily visualized in less than the5 min necessary to process the tissue (FIGS. 6D and D′), CT-b wasdetectable at the NMJ only several hours after injection (3-5 h). Thus,the dynamics of trafficking of CT-b and TTC receptors to active synapseare clearly different. However, after 5 h, the distribution obtained forCT-b was similar to GFP-TTC staining, as characterized by diffusestaining and patches having an extensive overlap of the stainingpatterns obtained with GFP-TTC, indicating a localization of the TTCprobe in lipid microdomains in motor nerve endings (FIG. 10B). Twentyfour hours after gastrocnemius injection, both toxins had beeninternalized since only few patches, most of them positive for bothtoxins, persisted at the NMJ (FIGS. 10B and C). At this time, GFP-TTCand CT-b staining were detected in the same motoneuron cell bodies inthe ventral horn of the spinal cord, but in different vesicularcompartments (FIG. 11). Taken together, these results indicate thatGFP-TTC used different lipid microdomains for neuronal binding and/orinternalization pathways than CT-b.

MATERIALS AND METHODS

[0127] Antibodies and Reagents.

[0128] Rabbit anti-GFP polyclonal antibodies was obtained fromInvitrogen (1:5000 dilution). Mouse monoclonal antibody against caveolin3 was from Transduction Laboratories (1:500). The monoclonalanti-neurofilament 200 (clone NE14) and the rabbit polyclonalantitroponin T were obtained from Sigma. AlexaFluor 594-conjugatedCholera toxin subunit B (CT-b); AlexaFluor 488-conjugatedgoat-antirabbit IgG, AlexaFluor 647-conjugated α-bungarotoxin (α-BTX)and RH414 were obtained from Molecular Probes. Cy3-conjugated goatanti-rabbit IgG and Cy3-conjugated rat anti-mouse IgG were from JacksonLaboratories. TRITC-conjugated α-bungarotoxin was obtained fromCalbiochem. The rabbit anti-TrkB (794) and the anti-p-Trk polyclonalantibody were obtained from SantaCruz. The monoclonal antibody againstsynaptic vesicle protein SV2, developed by K. Buckley, was obtained fromthe Developmental Studies Hybridoma Bank developed under the auspices ofthe NICHD and maintained by The University of Iowa, Department ofBiological Sciences, Iowa City. Monoclonal antibody against synapticvesicle synaptophysin protein was obtained from Chemicon. The goatanti-rabbit and anti-mouse IgG antibodies conjugated to horseradishperoxydase were obtained from Pierce as well as the SuperSignaldetection reagent. Recombinant neurotrophic factors rat CNTF; human NT3;human NT-4, human BDNF, human GDNF and purified mouse NGF 7S werepurchased from Alomone labs. Neurotrophic factors were prepared as stocksolutions (10 μg/ml) and kept in aliquots at −80° C.

[0129] In Vivo Intramuscular Injection

[0130] Experiments were performed in accordance with French and EuropeanCommunity guidelines for laboratory animal handling. Six-week-old Swissfemale mice were obtained from Charles River Breeding Laboratories.Intramuscular injections of β-gal-TTC, GFP-TTC fusion proteins, producedas previously described (Coen et al., 1997), or AlexaFluor594-conjugated CT-b were intramuscular injected into the gastrocnemiusmuscle or subcutaneously in the immediate vicinity of the Levator aurislongus (LAL) muscle on anesthetized mice. For fluorescencequantification, 25 μg of GFP-TTC fusion protein were injected in PBS in50 μl final volume. For immunodetection or biochemical extraction, 50 μgof GFP-TTC probe were used. When co-injections with neurotrophic factorswere performed, the volume injected was kept constant (50 μl). Forinjection in high K⁺, a physiological solution containing 60 mM KCl wasco-injected with the probe.

[0131]Botulinum type-A toxin injection.

[0132]Clostridium botulinum type-A toxin (BoTx/A) was injectedsubcutaneously as a single dose of 0.05 ml containing about 0.5 μg ofthe purified neurotoxin in the vicinity of the LAL muscle of femaleSwiss mice (body weight 24-27 g). 48 h after BoTx/A treatment, a timesufficient for inducing muscle paralysis in the LAL due to blockade ofneurotransmitter release (de Paiva et al., 1999), GFP-TTC (25 μg) wasinjected associated or not with BDNF (50 ng) in the vicinity of the LALmuscle. Mice were killed by intracardial injection of PFA 4% 30 minafter injection and LAL muscle harvested and processed for confocalanalysis.

[0133] In vitro analysis of GFP-TTC localization and confocalacquisition.

[0134] LAL muscles with their associated nerves were isolated fromfemale Swiss-Webster mice (20-25 g), killed by dislocation of thecervical vertebrae. LAL muscles were mounted in Rhodorsil^(R)-linedorgan baths (2 ml volume) superfused with a standard oxygenatedphysiological solution of the following composition (mM): NaCl 154; KCl5; CaCl₂ 2; MgCl₂ 1; HEPES buffer 5 (pH=7.4) and glucose 11. Muscleswere loaded for 45 min in the dark and at room temperature with both 25μg GFP-TTC and 30 μM of RH414, dissolved in standard solution or, forsynaptic vesicle recycling, in high K⁺ isotonic solution (with 60 mM KClreplacing 60 mM NaCl). Preparations were washed out of the GFP-TTC andRH414 dye, and rinsed several times with dye-free standard medium beforebeing imaged with a Leica TCS SP2 confocal laser scanning microscopesystem (Leica Microsystems, Germany) mounted on a Leica DM-RXA2 uprightmicroscope equipped with a ×40 water immersion lens (Leica, NA 0.8). Theconfocal system was controlled through Leica-supplied software runningon a Windows NT workstation.

[0135] Preparation of Detergent-Resistant Membrane (DRMs) Fractions andWestern Blot.

[0136] Preparation of detergent-resistant membrane fractions is one ofthe most widely used methods for studying lipid rafts. Two hours afterGFP-TTC injection (50 μg), mouse gastrocnemius muscle tissue washarvested, minced with scissors and homogenized in 2 ml of MES-bufferedsaline containing 1% (v/v) Triton X-100. Homogenization was carried outwith a Polytron tissue grinder. After centrifugation at low speed for 5min, supernatant was adjusted to 40% sucrose. A 5-30% linear sucrosegradient was formed above the homogenate and centrifuged at 39,000 rpmfor 18 h in a SW41 rotor. Then, 11-12 fractions of 1 ml were collectedfrom the top of the gradient and precipitated with 6.5% trichloroaceticacid in the presence of 0.05% sodium deoxycholate and washed with 80%cold acetone. Samples were analyzed by Western Blot after separating ona 4-15% SDS-PAGE followed by Western Blot. Membranes were probed firstwith polyclonal anti-GFP and monoclonal anti-caveolin 3 antibodies, andthen incubated with goat anti-rabbit IgGs and goat anti-mouse IgGsantibodies conjugated with horseradish peroxydase. The SuperSignal(Pierce) was used to visualize the reaction

[0137] Quantification of GFP-TTC Fluorescence Intensity at the NMJ.

[0138] After intracardiac perfusion and fixation, LAL muscles wereharvested, washed in PBS for 20 min, stained with TRITC-conjugateda-bungarotoxin (TRITC-a-BTX) (2 μg/ml) for 45 min at 37° C. in PBS andwashed twice in PBS. Images were acquired on an Axiovert 200M laserscanning confocal microscope (LSM-510 Zeiss; version 3.2) through a×40/1.2 water-immersion objective using LP560 and BP505-550 filters. Thepinhole aperture was set to 1 airy unit, and images were digitized at8-bit resolution into a 512×512 pixel array. To be able to compare theintensity of GFP staining between different experiments, laserillumination, photomultiplier gain in regard of linear response, andother acquisition variables were standardized. To quantify GFP-TTClocalization at the NMJ, series of “look-through” projection (of MIP:Maximum Intensity Projection) was generated. Images from each NMJ wereprocessed identically: NMJ surface area (in μm²) was determined byTRITC-a-BTX labeling and GFP fluorescence global intensity (sum of eachpixel intensity) was then measured only in this predefined area. Thisvalue, divided by the NMJ area yielded GFP fluorescence intensity persquare micrometer, which thus defined the fluorescence level expressedas arbitrary units. For each condition, ˜15 to 20 synapses werequantified and results were expressed as the mean±SD. Statisticalsignificance was defined as p<0.05 using a two-tailed t test. Eachexperiment was repeated at least two or three times.

[0139] Analysis of Spinal Cord.

[0140] 24 hours after β-gal-TTC or GFP-TTC and CT-b injection into thegastrocnemius muscle (50 μg each), mice deeply anesthetized wereperfused intracardially with 4% PFA. The spinal cord was harvested andembedded in Tissue Tek embedding media after overnight incubation in 25%sucrose in PBS 0.1 M. Longitudinal cryostat sections (30 μm thickness)were cut and mounted onto coated slides.

[0141] X-gal reaction.

[0142] X-gal reaction was performed as previously described (Coen etal., 1997).

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1 18 1 1600 DNA Clostridium tetani CDS (88)..(1476) 1 ggaaacagctatgaccatga ttacgccaag ctcgaaatta accctcacta aagggaacaa 60 aagctggagctcggtacccg ggccacc atg gtt ttt tca aca cca att cca ttt 114 Met Val PheSer Thr Pro Ile Pro Phe 1 5 tct tat tct aaa aat ctg gat tgt tgg gtt gataat gaa gaa gat ata 162 Ser Tyr Ser Lys Asn Leu Asp Cys Trp Val Asp AsnGlu Glu Asp Ile 10 15 20 25 gat gtt ata tta aaa aag agt aca att tta aattta gat att aat aat 210 Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn LeuAsp Ile Asn Asn 30 35 40 gat att ata tca gat ata tct ggg ttt aat tca tctgta ata aca tat 258 Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser ValIle Thr Tyr 45 50 55 cca gat gct caa ttg gtg ccc gga ata aat ggc aaa gcaata cat tta 306 Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala IleHis Leu 60 65 70 gta aac aat gaa tct tct gaa gtt ata gtg cat aaa gct atggat att 354 Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met AspIle 75 80 85 gaa tat aat gat atg ttt aat aat ttt acc gtt agc ttt tgg ttgagg 402 Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg90 95 100 105 gtt cct aaa gta tct gct agt cat tta gaa caa tat ggc acaaat gag 450 Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr AsnGlu 110 115 120 tat tca ata att agc tct atg aaa aaa cat agt cta tca atagga tct 498 Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile GlySer 125 130 135 ggt tgg agt gta tca ctt aaa ggt aat aac tta ata tgg acttta aaa 546 Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr LeuLys 140 145 150 gat tcc gcg gga gaa gtt aga caa ata act ttt agg gat ttacct gat 594 Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu ProAsp 155 160 165 aaa ttt aat gct tat tta gca aat aaa tgg gtt ttt ata actatt act 642 Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr IleThr 170 175 180 185 aat gat aga tta tct tct gct aat ttg tat ata aat ggagta ctt atg 690 Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly ValLeu Met 190 195 200 gga agt gca gaa att act ggt tta gga gct att aga gaggat aat aat 738 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu AspAsn Asn 205 210 215 ata aca tta aaa cta gat aga tgt aat aat aat aat caatac gtt tct 786 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln TyrVal Ser 220 225 230 att gat aaa ttt agg ata ttt tgc aaa gca tta aat ccaaaa gag att 834 Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro LysGlu Ile 235 240 245 gaa aaa tta tac aca agt tat tta tct ata acc ttt ttaaga gac ttc 882 Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu ArgAsp Phe 250 255 260 265 tgg gga aac cct tta cga tat gat aca gaa tat tattta ata cca gta 930 Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr LeuIle Pro Val 270 275 280 gct tct agt tct aaa gat gtt caa ttg aaa aat ataaca gat tat atg 978 Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile ThrAsp Tyr Met 285 290 295 tat ttg aca aat gcg cca tcg tat act aac gga aaattg aat ata tat 1026 Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys LeuAsn Ile Tyr 300 305 310 tat aga agg tta tat aat gga cta aaa ttt att ataaaa aga tat aca 1074 Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile LysArg Tyr Thr 315 320 325 cct aat aat gaa ata gat tct ttt gtt aaa tca ggtgat ttt att aaa 1122 Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly AspPhe Ile Lys 330 335 340 345 tta tat gta tca tat aac aat aat gag cac attgta ggt tat ccg aaa 1170 Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile ValGly Tyr Pro Lys 350 355 360 gat gga aat gcc ttt aat aat ctt gat aga attcta aga gta ggt tat 1218 Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile LeuArg Val Gly Tyr 365 370 375 aat gcc cca ggt atc cct ctt tat aaa aaa atggaa gca gta aaa ttg 1266 Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met GluAla Val Lys Leu 380 385 390 cgt gat tta aaa acc tat tct gta caa ctt aaatta tat gat gat aaa 1314 Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys LeuTyr Asp Asp Lys 395 400 405 aat gca tct tta gga cta gta ggt acc cat aatggt caa ata ggc aac 1362 Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn GlyGln Ile Gly Asn 410 415 420 425 gat cca aat agg gat ata tta att gca agcaac tgg tac ttt aat cat 1410 Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser AsnTrp Tyr Phe Asn His 430 435 440 tta aaa gat aaa att tta gga tgt gat tggtac ttt gta cct aca gat 1458 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp TyrPhe Val Pro Thr Asp 445 450 455 gag gga tgg aca aat gat taaacagattgatatgttca tgacatatgc 1506 Glu Gly Trp Thr Asn Asp 460 ccgggatcctctagagtcga cctcgagggg gggcccggta cccaattcgc cctatagtga 1566 gtcgtattacaattcactgg ccgtcgtttt acaa 1600 2 463 PRT Clostridium tetani 2 Met ValPhe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp 1 5 10 15 CysTrp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys Ser 20 25 30 ThrIle Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser Asp Ile Ser 35 40 45 GlyPhe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln Leu Val Pro 50 55 60 GlyIle Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu Ser Ser Glu 65 70 75 80Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp Met Phe Asn 85 90 95Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser 100 105110 His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met 115120 125 Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys130 135 140 Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu ValArg 145 150 155 160 Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn AlaTyr Leu Ala 165 170 175 Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp ArgLeu Ser Ser Ala 180 185 190 Asn Leu Tyr Ile Asn Gly Val Leu Met Gly SerAla Glu Ile Thr Gly 195 200 205 Leu Gly Ala Ile Arg Glu Asp Asn Asn IleThr Leu Lys Leu Asp Arg 210 215 220 Cys Asn Asn Asn Asn Gln Tyr Val SerIle Asp Lys Phe Arg Ile Phe 225 230 235 240 Cys Lys Ala Leu Asn Pro LysGlu Ile Glu Lys Leu Tyr Thr Ser Tyr 245 250 255 Leu Ser Ile Thr Phe LeuArg Asp Phe Trp Gly Asn Pro Leu Arg Tyr 260 265 270 Asp Thr Glu Tyr TyrLeu Ile Pro Val Ala Ser Ser Ser Lys Asp Val 275 280 285 Gln Leu Lys AsnIle Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser 290 295 300 Tyr Thr AsnGly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr Asn Gly 305 310 315 320 LeuLys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser 325 330 335Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser Tyr Asn Asn 340 345350 Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn 355360 365 Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu370 375 380 Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr TyrSer 385 390 395 400 Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser LeuGly Leu Val 405 410 415 Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro AsnArg Asp Ile Leu 420 425 430 Ile Ala Ser Asn Trp Tyr Phe Asn His Leu LysAsp Lys Ile Leu Gly 435 440 445 Cys Asp Trp Tyr Phe Val Pro Thr Asp GluGly Trp Thr Asn Asp 450 455 460 3 1392 DNA Clostridium tetani 3atggtttttt caacaccaat tccattttct tattctaaaa atctggattg ttgggttgat 60aatgaagaag atatagatgt tatattaaaa aagagtacaa ttttaaattt agatattaat 120aatgatatta tatcagatat atctgggttt aattcatctg taataacata tccagatgct 180caattggtgc ccggaataaa tggcaaagca atacatttag taaacaatga atcttctgaa 240gttatagtgc ataaagctat ggatattgaa tataatgata tgtttaataa ttttaccgtt 300agcttttggt tgagggttcc taaagtatct gctagtcatt tagaacaata tggcacaaat 360gagtattcaa taattagctc tatgaaaaaa catagtctat caataggatc tggttggagt 420gtatcactta aaggtaataa cttaatatgg actttaaaag attccgcggg agaagttaga 480caaataactt ttagggattt acctgataaa tttaatgctt atttagcaaa taaatgggtt 540tttataacta ttactaatga tagattatct tctgctaatt tgtatataaa tggagtactt 600atgggaagtg cagaaattac tggtttagga gctattagag aggataataa tataacatta 660aaactagata gatgtaataa taataatcaa tacgtttcta ttgataaatt taggatattt 720tgcaaagcat taaatccaaa agagattgaa aaattataca caagttattt atctataacc 780tttttaagag acttctgggg aaacccttta cgatatgata cagaatatta tttaatacca 840gtagcttcta gttctaaaga tgttcaattg aaaaatataa cagattatat gtatttgaca 900aatgcgccat cgtatactaa cggaaaattg aatatatatt atagaaggtt atataatgga 960ctaaaattta ttataaaaag atatacacct aataatgaaa tagattcttt tgttaaatca 1020ggtgatttta ttaaattata tgtatcatat aacaataatg agcacattgt aggttatccg 1080aaagatggaa atgcctttaa taatcttgat agaattctaa gagtaggtta taatgcccca 1140ggtatccctc tttataaaaa aatggaagca gtaaaattgc gtgatttaaa aacctattct 1200gtacaactta aattatatga tgataaaaat gcatctttag gactagtagg tacccataat 1260ggtcaaatag gcaacgatcc aaatagggat atattaattg caagcaactg gtactttaat 1320catttaaaag ataaaatttt aggatgtgat tggtactttg tacctacaga tgagggatgg 1380acaaatgatt aa 1392 4 49 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 ccccccgggc caccatggtt ttttcaacac caattccattttcttattc 49 5 18 DNA Artificial Sequence Description of ArtificialSequence Primer 5 ctaaaccagt aatttctg 18 6 25 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 aattatggac tttaaaagat tccgc25 7 24 DNA Artificial Sequence Description of Artificial SequencePrimer 7 ggcattataa cctactctta gaat 24 8 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 8 aatgccttta ataatcttgatagaaat 27 9 41 DNA Artificial Sequence Description of ArtificialSequence Primer 9 ccccccgggc atatgtcatg aacatatcaa tctgtttaat c 41 10 24DNA Artificial Sequence Description of Artificial Sequence Primer 10ctgaatatcg acggtttcca tatg 24 11 40 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 11 ggcagtctcg agtctagacc atggctttttgacaccagac 40 12 20 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide linker 12 catgactggg gatccccagt 20 1324 DNA Artificial Sequence Description of Artificial Sequence Primer 13tatgataaaa atgcatcttt agga 24 14 37 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 14 tggagtcgac gctagcagga tcatttgtccatccttc 37 15 8 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide linker 15 gctagcgc 8 16 17 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide linker 16 gatatcggcg cgccagc 17 17 17 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotidelinker 17 tggcgcgccg atatcgc 17 18 14 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide linker 18tcgatggcgc gcca 14

What is claimed is:
 1. A method for in vivo delivery of a desiredcomposition into human or animal central nervous system (CNS) or spinalcord, wherein the method comprises administering to the human or animala composition comprising a non-toxic, proteolytic fragment of tetanustoxin (TT) in association with at least a molecule having a biologicalfunction and said composition is capable of in vivo retrograde axonaltransport and transynaptic transport into the CNS or the spinal cord ofthe human or animal and of being delivered at different areas of the CNSor the spinal cord.
 2. The method according to claim 1, wherein thecomposition is administered into a muscle.
 3. The method according toclaim 1, wherein the composition is administered into a muscle in thevicinity of a neuromuscular junction.
 4. The method according to claim1, wherein the muscle is selected in relation with the desired area ofthe CNS or spinal cord.
 5. The method according to claim 1, wherein thecomposition is administered into neuronal cells.
 6. The method accordingto claim 1, wherein the composition comprises a non-toxic, proteolyticfragment of tetanus toxin (TT) comprising a fragment C and a fragment Bor a fraction thereof of at least 11 amino acid residues in associationwith at least a molecule having a biological function selected from thegroup consisting of a protein for compensation or modulation offunctions under the control of the CNS or the spinal cord or modulationof functions in the CNS or the spinal cord or a protein to be deliveredby gene therapy expression system to the CNS or the spinal cord.
 7. Themethod according to claim 1, wherein the composition comprises anon-toxic, proteolytic fragment of tetanus toxin (TT) comprising afragment C and a fragment B or a fraction thereof of at least 11 aminoacid residues and a fraction of a fragment A devoid of its toxicactivity corresponding to the proteolytic domain having a zinc-bindingmotif located in the central part of the chain between amino acids 225and 245 in association with at least a molecule having a biologicalfunction selected from the group consisting of protein for thecompensation or the modulation of functions under the control of the CNSor the spinal cord or protein to be delivered by gene therapy expressionsystem to the CNS or the spinal cord.
 8. The method according to claim 6or claim 7, wherein the molecule is selected from the group consistingof protein SM, BDNF (Brain-derived neurotrophic factor), NT-3(Neurotrophin-3), NT-4/5, GDNF (Glial cell-line-derived neurotrophicfactor), IGF (Insulin-like growth factor), PNI (protease nexin I), SPI3(Serine Protease Inhibitor protein), ICE (Interleukin-1β convertingenzyme), Bcl-2, GFP (green fluorescent protein), endonucleases likeI-SceI or CRE, antibodies, or drugs specifically directed againstneurodegenerative diseases such as latero spinal amyotrophy (LSA). 9.The method according to claim 8, wherein the composition comprises acombination of at least two of said molecules.
 10. The method accordingto claim 8, wherein the molecule is located upstream from the fragmentof tetanus toxin.
 11. The method according to claim 8, wherein themolecule is located downstream from the fragment of tetanus toxin. 12.The method according to claim 1, which comprises administering to thehuman or animal a vector containing nucleotides encoding thecomposition, wherein the vector is capable of in vivo expression in amuscle and this product is capable of migrating to the CNS or spinalcord.
 13. The method according to claim 12, wherein said vectorcomprises a promoter and an enhancer capable of expressing thenucleotides contained in said vector in the muscle.
 14. The methodaccording to claim 13, wherein said vector is the plasmid pCMV-LacZ-TTCwhich has been deposited at the C.N.C.M. on Aug. 12, 1997, under theregistration number 1-1912.
 15. The method according to claim 12 or 13,wherein said vector is administered into the muscle.
 16. The methodaccording to claim 12 or 13, wherein the molecule is a nucleotideencoding for a protein or a polypeptide linked chemically to thefragment of tetanus toxin and being transported and expressed directlyin neurons.
 17. A hybrid fragment of tetanus toxin comprising a fragmentC and a fragment B or a fraction thereof of at least 11 amino acidresidues capable of transferring in vivo a protein, a peptide, or apolynucleotide through a neuromuscular junction and at least onesynapse.
 18. A hybrid fragment of tetanus toxin comprising a fragment Cand a fragment B or a fraction thereof of at least 11 amino acidresidues and a fraction of a fragment A devoid of its toxic activitycorresponding to the proteolytic domain having a zinc-binding motiflocated in the central part of the chain between amino acids 225 and 245capable of transferring in vivo a protein, a peptide or a polynucleotidethrough a neuromuscular junction and at least one synapse.
 19. An aminoacid variant fragment having the same properties as the hybrid fragmentof tetanus toxin according to claims 17 or
 18. 20. A polynucleotidevariant fragment capable of hybridization under stringent conditionswith the natural tetanus toxin sequence.
 21. A composition containing anactive molecule in association with a hybrid fragment of tetanus toxinaccording to claims 17 or 18 or with an amino acid variant fragmentaccording to claim
 16. 22. The composition according to claim 21,wherein the active molecule is selected from the group consisting ofprotein SMN, BDNF (Brain-derived neurotrophic factor), NT-3, NT-4/5,GDNF (Glial cell-line derived neurotrophic factor), IGF (Insulin-likegrowth factor), PNI (protease nexin I), SP13 (Serine Protease Inhibitorprotein), ICE, Bcl-2, GFP (green fluorescent protein), endonucleaseslike I-SceI or CRE, antibodies or drugs specifically directed againstneorodegenerative diseases such as latero spinal amyotrophy (LSA). 23.The composition according to claim 21, wherein the active molecule is apolynucleotide encoding a protein or a polypeptide with a promotercapable of expression in neurons, and optionally an enhancer.
 24. Avector comprising a promoter capable of expression in muscle cells andoptionally an enhancer, a nucleic acid sequence coding for the fragmentof tetanus toxin according to claims 17 or 18 or with an amino acidvariant fragment according to claim 19 associated with a polynucleotidecoding for a protein or a polypeptide.
 25. A method of treatment of apatient or an animal affected with CNS or spinal cord disease, whichcomprises delivering a composition according to claims 21, 22, or 23 tothe patient or animal in an amount effective for treatment of the CNS orspinal cord disease.
 26. A method of treatment of a patient or an animalaffected with CNS or spinal cord disease, which comprises delivering avector according to claim 24 to the patient or animal in an amounteffective for treatment of the CNS or spinal cord disease.
 27. Themethod according to claim 1, which comprises administering to the humanor animal a cell or a vector containing nucleotides encoding thecomposition, wherein the cell or vector is capable of in vivo expressionin neuronal cells or precursor of neuronal cells and wherein said cellis reimplanted into the CNS or spinal cord.
 28. The method according toclaim 27 wherein said cell or vector comprises a promoter and anenhancer capable of expressing the nucleotides contained in said cell inneuronal cells or precursors of neuronal cells.
 29. The method accordingto claim 27 or 28 wherein the molecule is a nucleotide encoding for aprotein or a polypeptide linked chemically to the fragment of tetanustoxin and being expressed directly in neurons.
 30. The method accordingto claim 27 or 28 wherein the molecule is a nucleotide encoding for aprotein or a polypeptide linked chemically to the fragment of tetanustoxin and being expressed directly in neurons.
 31. A cell or vectorcomprising a promoter capable of expression in neuronal cells orprecursors of neuronal cells and optionally an enhancer, a nucleic acidsequence coding for the fragment of tetanus toxin according to claims 17or 18 or with an amino acid variant fragment according to claim 19associated with a polynucleotide coding for a protein or a polypeptide.32. A method of modulating the transport in a neuron of a tetanus toxinor a fusion protein comprising a fragment C of the tetanus toxin,wherein the method comprises administering to the neuron a TrkB receptoragonist or a TrkB receptor antagonist in an amount sufficient tomodulate the neuronal transport of the tetanus toxin or the fusionprotein.
 33. The method according to claim 32, wherein the TrkB receptoragonist is administered, thereby increasing the internalization of thetetanus toxin or fusion protein at a neuromuscular junction.
 34. Themethod according to claim 33, wherein the TrkB receptor agonist is aneurotrophic factor that activates a TrkB receptor.
 35. The methodaccording to claim 34, wherein the neurotrophic factor is a BrainDerivated Neurotrophic Factor or a Neurotrophin
 4. 36. The methodaccording to claim 33, wherein the TrkB receptor agonist is an antibodythat binds to a TrkB receptor, thereby activating the TrkB receptor. 37.The method according to any one of claims 35 or 36, wherein theinternalization of the fusion protein at the neuromuscular junction isincreased.
 38. The method according to claim 32, wherein the TrkBreceptor antagonist is administered, thereby decreasing theinternalization of the tetanus toxin or fusion protein at aneuromuscular junction.
 39. The method according to claim 38, whereinthe TrkB receptor antagonist is an antibody that binds to a TrkBreceptor agonist, thereby reducing activation of a TrkB receptor. 40.The method according to claim 39, wherein the TrkB receptor agonist is aneurotrophic factor that activates a TrkB receptor.
 41. The methodaccording to claim 40, wherein the neurotrophic factor is a BrainDerivated Neurotrophic Factor or a Neurotrophin
 4. 42. The methodaccording to claim 42, wherein the internalization of the tetanus toxinat the neuromuscular junction is decreased.
 43. The method according toclaim 40, wherein the neurotrophic factor is administered concurrentlywith the fusion protein.
 44. A method of modulating the transport in aneuron of a tetanus toxin or a fusion protein comprising a fragment C ofthe tetanus toxin, wherein the method comprises administering to theneuron a GFRα/cRET receptor agonist or a GFRα/cRET receptor antagonistin an amount sufficient to modulate the neuronal transport of thetetanus toxin or the fusion protein.
 45. The method according to claim44, wherein the GFRα/cRET receptor agonist is administered, therebyincreasing the internalization of the tetanus toxin or fusion protein ata neuromuscular junction.
 46. The method according to claim 45, whereinthe GFRα/cRET receptor agonist is a neurotrophic factor that activates aGFRα/cRET receptor.
 47. The method according to claim 46, wherein theneurotrophic factor is a Glial-Derived Neurotrophic Factor.
 48. Themethod according to claim 44, wherein the GFRα/cRET receptor agonist isan antibody that binds to a GFRα/cRET receptor, thereby activating theGFRα/cRET receptor.
 49. The method according to any one of claims 46 or47, wherein the internalization of the fusion protein at theneuromuscular junction is increased.
 50. The method according to claim44, wherein the GFRα/cRET receptor antagonist is administered, therebydecreasing the internalization of the tetanus toxin or fusion protein ata neuromuscular junction.
 51. The method according to claim 50, whereinthe GFRα/cRET receptor antagonist is an antibody that binds to aGFRα/cRET receptor agonist, thereby reducing activation of a GFRα/cRETreceptor.
 52. The method according to claim 51, wherein the GFRα/cRETreceptor agonist is a neurotrophic factor that activates a GFRα/cRETreceptor.
 53. The method according to claim 52, wherein the neurotrophicfactor is a Glial-Derived Neurotrophic Factor.
 54. The method of claim53, wherein the internalization of the tetanus toxin at theneuromuscular junction is decreased.
 55. The method according to claim47, wherein the neurotrophic factor is administered concurrently withthe fusion protein.
 56. A composition, comprising a TrkB receptoragonist and a fusion protein comprising a fragment C of the tetanustoxin fused to a second protein.
 57. The composition according to claim56, wherein, the TrkB receptor antagonist is a neurotrophic factor thatactivates a TrkB receptor.
 58. The composition according to claim 57,wherein the neurotrophic factor is a Brain Derivated Neurotrophic Factoror a Neurotrophin
 4. 59. A composition, comprising a GFRα/cRET receptoragonist and a fusion protein comprising a fragment C of the tetanustoxin fused to a second protein.
 60. The composition according to claim59, wherein, the GFRα/cRET receptor antagonist is a neurotrophic factorthat activates a GFRα/cRET receptor.
 61. The composition according toclaim 60, wherein the neurotrophic factor is Glial-Derived NeurotrophicFactor.
 62. A method of detecting an effect of a compound on neuronaltransport, comprising administering to a neuron the compound and afusion protein comprising a fragment C of the tetanus toxin fused to asecond protein, wherein the second protein is encoded by a reportergene, and detecting the second protein to determine the effect of thecompound on neuronal transport.
 63. The method according to claim 62,wherein the compound is a neurotophic factor.
 64. A method of screeningfor a compound that reduces or prevents transport of a tetanus toxin ina neuron, comprising administering to the neuron the compound and afusion protein comprising a fragment C of the tetanus toxin fused to asecond protein, wherein the second protein is encoded by a reportergene, detecting the second protein, and selecting the compound thatreduces or prevents the neuronal transport of the fusion protein. 65.The method according to claim 64, wherein the second protein is detectedat a neuromuscular junction.